In this study we used dox, a mainstream chemotherapeutic agent, which is inherently fluorescent, as a model drug to demonstrate its cellular uptake significance in our TSDR model. Dox is known to enter a cell, intercalate to DNA and block topoisomerase II. The cell’s ability to proliferate is then strictly conserved to a number of intracellular dox molecules attained by DNA (28). The question is if this intracellular drug indwelling in tissues for years (and recycling locally) can keep controlling a minimal residual disease. For more than five decades, dox is widely used as a front-line therapy for several different malignancies. However, its role in controlling residual micrometastatic diseases (if any) is not completely understood. Chemotherapy does not only spare some viable tumor cells but also results in a long-term tissue persistence of injected drug. The cellular bound drug and its active metabolites definitely undergo some gradual transformation while indwelling in tissues. The characteristic feature of this transformation and its effect on tumor and host tissue is not studied. It can feature decreased or increased antitumor (and dormancy maintaining) activity. Our dox-dgr samples showed obvious visual change but we analyzed only some of its physicochemical properties. Spontaneous aggregation of dox is known to induce a decrease of the fluorescence quantum yield attributed to the formation of non-fluorescent dimers (27) Self-association is a process that competes with binding to DNA and formation of hetero complexes (27, 29).
Tumor cells loaded with dox following exposure to 10 µg/ml, lost their ability to resume proliferation in DBA/2 mice when injected ip. Exposure of cells to lower doses of dox did not result in any recurrence delay. The SL2 cell viability (> 90%) after dox exposure of all samples before their ip implantation in mice was sufficient to produce tumors. As little as 100 cells were reported to be enough to result in ascitic SL2 lymphoma growth and kill the mice (24). The rate of proliferation and aggressiveness of SL2 line is notable: the growth fraction of an SL2 tumor on day 4 after implantation is 79.2% (30). So just one viable cell out of each 5000 cells implanted (0.02% viability) should have been sufficient enough to assure tumor formation and growth. We hypothesized that gradual extrusion of dox from the tumor cells should have resulted in the tumor escaping from the dormancy state attained after the initial drug cellular accumulation. A tumor exposed to 10 µg/ml dox for 30 min, was empirically determined to be sufficient enough to stop tumor proliferation. Subsequently, a pharmacokinetic study was conducted to clarify how this exposure (in terms of dox concentration and AUC) compares to plasma dox concentration detectable after a 15 mg/kg dox injection. The highest plasma dox concentration was reached at 5 minutes post injection, and was only 1.33 µg/ml. It might be questionable if our TSDR model is relevant to an in vivo animal or clinical conditions. That is because we failed to demonstrate that maximum plasma dox concentration ever reached 10 µg/ml in mice following a high dose of dox administration. However, several facts are to be taken into consideration: 1) tissue and cells in mice following the injection were exposed to detectable dox plasma levels considerably longer than 30 min; dox in the plasma of treated DBA/2 mice was detectable for at least 72 hr, whereas in vitro exposure conditions lasted just 30 min, 2) the AUC0 − 0.5 of TSDR model exposure was 5.0 (µg•h/ml) whereas AUC0 − 72 of dox in mice plasma following a 15 mg/kg iv. injection was 3.6 (µg•h/ml); the same exposure dose (10 µg/ml) can be compared to dox uptake investigations done by others; For instance, the duration of 10 µg/ml exposure of 5 days was reported (26); 4) apparently, the short tissue exposure to dox is more relevant to clinical situations than AUC (31), 5) the sufficient cellular uptake of dox is already seen following 5 sec of exposure and might play a role in tumor growth control (32). It is technically impossible to test if, at a 5 sec time interval, the plasma levels are or are not reaching 10 µg/ml following dox iv administration. The max plasma concentration in humans was found to be in the vicinity of 1–4 µg/ml after the administration of common therapeutic doses (33). However, major exposure of cells and tissue to dox in humans is found in the terminal elimination phase where plasma concentrations are around 0.05 µg/ml and continues to persist for at least 48 hr (34). Not only dox, but also its active metabolites, are found in significant amounts in human plasma and are contributing to its antiproliferative effects.
The intracellular dox tend to be retained in tissues for months and years. The accidentally extravasated dox was found to be present in significant amounts in tissue adjacent to the extravasation site for 5 months (8). Tissue uptake of dox is hundreds of times higher than the maximum plasma concentration (7). We expected that dox-dgr can partly simulate the tissue indwelling of dox and should have retained some antiproliferative capacity. The short term (21 day) dox 37°C exposure did not reveal any absorbance or fluorescence spectrum alteration in an aqueous solution or cellular nuclei (data not shown). However, at medium term (120 days), the exposure to dox-dgr lost its antiproliferative capacity and was not capable of inhibiting tumor cell recurrence in our TSDR model. Upon HPLC analysis, the residual dox peak was still noticeable, although it exhibited significant quantitative decline. The SL2 cells prepared for ip injection revealed identical intracellular fluorescence spectra for dox and dox-dgr as judged by confocal microscopy. Fluorescence intensity and uptake patterns however, were dramatically altered in dox-dgr exposed samples as compared to fresh dox samples. There was no preferential nuclear accumulation in samples exposed to dox-dgr, which was a characteristic feature of fresh dox uptake. This raises the question if nuclear dox binding capacity is not undergoing transformation following long term body indwelling of a drug. In fact, cellular binding patterns might have a direct link to dox’s therapeutic efficacy. For instance, dox cytotoxicity in a clonogenicity assay was shown to be highly dependent on cellular accumulation patterns (35). Internalized tightly bound cellular dox was the only active form of drug eliciting cytotoxic effects, as reported in this study. The tightly bound dox was not very prone to outward efflux. However, 90% of intracellular dox could be attributed to a loose bound category demonstrated in this investigation.
Dox is known to enter the cell by passive diffusion, but at lower concentrations much of it is taken up rapidly by high-affinity binding sites (36). At higher concentrations, these binding sites are saturated and additional drug taken up by the cell remains unbound in the intracellular space. It is not clear if this intracellular unbound dox can play the role of lose bound fraction and be prone to a rapid extrusion upon IP implantation in our TSDR model.
Our findings might be questioning the supposition that long term control of micrometastatic disease is elicited through direct antitumor effects of cellular bound drug. Our assumption is based upon the fact that long term simulation of indwelling drug failed to demonstrate residual antiproliferative activity. However, this does not rule out the possibility that tissue bound dox is still involved in suppressing tumor regrowth at least for several months following discontinuation of chemotherapy. This temporal effectiveness of dormancy control may alter the tumor microenvironment and activate some other host mediated tumor control mechanisms (i.e. immune surveillance). Our findings do not rule out the possibility that some marginal antiproliferative capacity of dox-dgr was still preserved. The WBC nadir assay of dox (15 mg/kg, iv) revealed a dramatically higher antiproliferative capacity for fresh dox compared to the dox-dgr. This rather high Dox concentration was chosen to achieve a drug in vivo exposure in the vicinity of exposure explored in TSDR model at 10 µg/ml in vitro. However, the relative weakness of dox-dgr in nadir assay might simply be due to a smaller amount of active dox fraction within a whole dox-dgr vial. Similarly, CCK-8 cytotoxicity assay might be simply a result of less intact drug remaining in a test sample when testing dox-dgr. The HPLC analysis demonstrated that dox-dgr retains only 16.8% of the initial dox amount after 120 days at 37°C. This means the 15 mg/kg of dox-dgr would correspond to roughly 2.5 mg/kg of fresh dox. Interestingly, 2.5 mg/kg of fresh dox injected iv into DBA/2 mice did not show significant WBC decline at a 48-hour time point (data not shown). Even fresh dox tends to form pharmacologically inactive dimers in a vial at room temperature. Some 47% of the fresh dox can be attributable to these spontaneously forming byproducts (29). We cannot rule out the possibility that DNA bound dox might retain even more antiproliferative capacity than the dox kept at body temperature in the original vial. In theory DNA intercalated dox molecules should not be forming inactive aggregates in vivo.
Dox was rather extensively studied in the models of forced degradation under the conditions of hydrolysis (water, acid and alkali), peroxide oxidation, dry heat (short duration at 80°C), photolysis and microwave irradiation (37, 38). To our knowledge, our study is the first to report on the degradation of dox stored at body temperature for the duration of 365 days. We were not specifically identifying the byproducts related to the dox decay, we instead tested the antitumor and myelotoxic properties of a whole dox-dgr product.
The dormancy/recurrence model developed by others with SU159 cells (25) explored a lower dox concentration (0.5 µg/ml) than ours. However, substantially longer exposure time (96 hr) was used to demonstrate dormancy in vitro. This can be converted into AUC0 − 96 48 µg/ml which is significantly higher than the one used in our TSDR model. Interestingly this duration of exposure of dox with SU159 cells was considered to be short. Notably, the maximum plasma concentration of dox results in a peak concentration higher than 1.0 µg/ml in humans (33). In the model proposed by El-Kareh et al. (31), the dox mediated cell kill was demonstrated to be dependent on the peak concentrations rather than the time integral of concentration. We hypothesized that 96 hr in vitro exposure (25) might not entirely duplicate the in vivo situation where dox plasma levels are barely detectable. The in vitro model is for cells in culture, where oxygenation, pH, cell density, extracellular dox clearance, drug metabolism in liver and elimination in bile or urine are generally different than conditions in vivo. An empirically determined short exposure procedure which was blocking successful tumor implantation can help to better understand the mechanisms of local cytotoxic drug effects. For instance, dox at a dose of 1000 µg/ml for 60–120 min is used by intravesical instillation for the treatment of superficial bladder carcinoma. This is a 100 times higher concentration than explored in our study. In addition, the intravesical instillation is sometimes repeated with an interval of 1 week to 1 month, depending on whether the treatment is therapeutic or prophylactic. A local injection at the maximum safe dose of dox (1000 µg in the upper lid and 1500 µg in the lower lid) was used for the treatment of blepharospasm (39). The cumulative dose in this study was even higher – 4000 µg of dox per lid. Local implantable surgical devices, such as biodegradable mesh, are occasionally patented to claim therapeutic efficacy of anticancer drugs including dox (40). A dose of 10 µg/ml for 30 min definitely remains in the safe zone for a vesicant drug such as dox when applied locally. The effective concentration of dox or any other cytotoxic drug can be additionally investigated with the help of our TSDR model. Dox controlled release via biodegradable ureteral stent (41) is an example where this model could be explored for a rapid pre-screening to determine an effective concentration range for local use.
The idea that long term tumor exposure to suboptimal doses is breeding genetically resistant cells is just one of the popular premises. This assumption is based mostly on experiments in vitro. It is a well-known fact that resistant lines can be continuously passed in media containing a cytotoxic drug and losing no proliferative capacity. The resistance in humans is not necessarily mimicking this effect. Drug intracellular presence is the most obvious feature currently known to restrain cells from resumed proliferation. Notably, the primary resistance in humans and laboratory animals might be explained for instance by inoculum effect (42) or abundance of tumor cells (43). Multiple other non-genomic resistance factors were described: non-genetic plasticity (44), drug gradients in relation to blood vessels (45), specificities of an extracellular matrix (46), and an abundance of cellular debris (47). All of these conditions are not easy to replicate in models relevant to clinical conditions. Our TSDR model is an attempt to elucidate one of the aspects of primary resistance and dormancy, which is the link between tissue retained drug and tumor recurrence.
Our study is not without certain limitations. The immunological mechanisms rather than drug efflux might have played an important role in tumor rejection in TSDR animals. The failure of animals to reject the secondary rechallenge does not indicate the absence of a strong syngeneic sensibilization at the initial in vivo phase of TSDR. The rechallenge time point was quite remote – 75 days and according to 60 days long-term survival criteria could not be done much earlier. The selected rechallenge tumor dose was potentially too high (equivalent to the initial phase of TSDR). The SL2 tumor is highly aggressive and is known to be engrafted ip at a dose as low as 10–100 cells per animal. The SL2 is definitely an immunogenic tumor and it was demonstrated by several investigators (including our group) that SL2 can be used as a classical model of “concomitant immunity” (48). However, the specific concomitant immunity phenomenon observed in SL2 model is transient and short lived (49). The nonspecific phase of concomitant immunity in SL2 model might be simply explained by antimitotic factor release from primary tumor. The duration of tumor antigen exposure adds to the complexity of immune mechanism understanding. Interestingly, murine T-cell lymphoma EL4 bearing mice became resistant to the late tumor rechallenge following successful dox plus interleukin 2 treatment (50). It means that more mechanisms involving memory cell generation in syngeneic tumor dormancy are yet to be elucidated.