For the development and evaluation of novel anti-cancer therapies, solely tumor growth measurements often miss initial molecular tumor responses. Therefore molecular imaging of tumor response with FDG-PET has advanced into experimental study designs allowing to follow up therapeutic effects in dedicated tumor models. Nevertheless influx of inflammatory cells can lead to underestimation of the therapeutic effects and allows only an indirect measurement of tumor viability. In this perspective the visualization of the number of viable tumor cells with BLI might allow an accurate, highly sensitive and easy technique to monitor therapeutic effects in preclinical animal models. Nevertheless BLI is a 2D imaging technique that is surface-weighted while FDG-PET measures metabolism in three dimensions. In the current study we evaluated BLI and compared results with FDG-PET and changes in tumor size. We chose to evaluate two types of treatment agents; cyclophosphamide, a standard chemotherapeutic, and temsirolimus, inducing mainly cell cycle arrest.
BLI analysis revealed largely augmented BLI signal intensities early after treatment which were considerably higher when compared to non-treated animals scanned on this time point. This effect seemed to be higher for cyclophosphamide compared to temsirolimus therapy. Surprisingly only few studies reported an increased BLI signal early after therapy which were suggested to be due to metabolic changes induced by the therapy such as changes in membrane permeability, ATP elevation due to stress response or apoptosis [19
], degradation of the membrane integrity or transient changes in transcription [14
]. The induction of apoptosis was more pronounced for cyclophoshpamide when compared to temsirolimus which may in part explain the higher BLI signal. Additionally cyclophoshamide is described to induce DNA repair early after therapy which also induces higher metabolic activity of cells [23
]. Besides metabolic changes following therapy, the transcription of the promoter may be altered following toxic treatment. This was already described for the cytomegalovirus (CMV) promoter demonstrating BLI increase up to 3 times control values following cyclophosphamide therapy [14
]. No such activation of the spleen focused forming virus (SFFV), used in the current study, has yet been described.
In a recent publication by M Keyaerts et al., the effect of protein concentrations has been demonstrated to have vast influence on BLI signal. In this regards, toxic therapies such as doxorubicin can induce hypoalbuminemia resulting in an increased BLI signal [15
]. Although hypoalbuminemia has not been described for cyclophosphamide and temsirolimus, these toxic agents can influence plasma protein concentrations and result in altered substrate binding leading to an increased BLI signal.
Overall, several processes may explain the increased BLI signal and most likely a combination resulted in the increased BLI levels. The specific assessment of the processes involved in this interplay was beyond the scope of this project and needs extensive in vitro and in vivo research. Unfortunately these results suggest that metabolic or molecular changes following therapy, not necessarily correlated to viability, can influence the BLI signal early after therapy which should be considered when used for response assessment.
Also on later time points, BLI failed to depict a decreased amount of viable cells earlier than tumor shrinkage. The BLI signal decreased significantly when tumor volumes were largely reduced for temsirolimus on day 9 (-47 ± 7% Volcalip) and for cyclophosphamide on day 14 (-85 ± 4% Volcalip). As a result BLI was not able to depict a reduced amount of viable cells while an undoubted reduction in the amount of viable cells was measured by caliper (Volcalip) on day 4 for temsirolimus and day 7 for cyclophosphamide. This can be explained by the fact that the BLI flare was not restricted to the early time points following therapy while t he metabolic changes may hold true for the still viable cells and effects such as decreased plasma membrane proteins can sustain an increased BLI signal. Additionally other factors such as tumor size, shape and vasculature can influence the penetration of the photons and become important on later time points following therapy. Tumor size reduction enhances the penetration of photons which may result in an overestimation of the number of viable cells.
From this perspective, we suggest that BLI can be useful to monitor therapy but requires sequential scans to obtain the complete BLI response profile. Additionally further experiments will be necessary to elucidate the exact mechanisms of BLI flare which may hold promises and new perspectives to follow therapy induced changes. For instance, the BLI flare may be related to apoptosis which may serve as an early indication of response reflected as therapy-induced apoptosis.
Nevertheless, our results together with other reports, suggest that BLI will not beat FDG-PET as an early predictive marker for response. FDG-PET was able to depict decreased metabolism before the tumor had reduced in size, which offers possibilities for early response assessment. Additionally FDG-PET is able to measure small metabolic changes following therapy and allows to follow-up the dynamics of therapeutic agents on the cell metabolism. In our study the FDG uptake decreased immediately in both treatment groups being related to the immediate induction of apoptosis. Nevertheless the effect of cyclophosphamide was more pronounced with more apoptosis and almost complete disappearance of the tumors as depicted in the example (). On the other hand the effect of temsirolimus on tumor survival was fast (fast induction of apoptosis and fast shrinkage of tumor) while it did not result in complete disappearance of the tumor. This was not reflected in the caliper measurement while FDG-PET suggests an increased FDG uptake on day 14 following temsirolimus which indicates regrowth of the tumor ().
The effect of inflammation on the FDG uptake was small in this animal model. Previous studies in the same animal model demonstrated influx of inflammatory cells between day 7 and day 9 following cyclophosphamide therapy [5
]. Although no increased FDG uptake was seen on these time points we observe an FDG plateau between day 4 and day 7 following cyclophosphamide while tumor size decreases gradually. Unlike cyclophoshamide, temsirolimus does not induce major influx of inflammatory cells [23
]. Overall, this study adds more value to the use of FDG-PET for pre-clinical assessment of molecular response allowing to evaluate the effectiveness and function of novel anti-cancer therapies.