In order to explore if a range of conventional anti-cancer drugs would impact on the cellular uptake of FDG, we utilized multicellular tumour spheroids, treated with the drugs and after a few days of treatment evaluated the uptake of FDG. Two different breast cancer cell lines were selected with different characteristics. BT474 cells have up-regulated mRNA and HER2/neu tyrosine kinase-linked receptor protein in comparison to MCF7, while the expression of the estrogen receptor alpha is known to be up-regulated in MCF7 cells. These two cell-lines are assumed to be representative for a variety of breast cancers.
Although growing cells as MTS is more laborious than monolayer, MTS cells more closely mimic the real biological environment by providing cell-to-cell adhesion and signalling. This gives a more relevant picture of the drug effects by including limitations in penetration, distribution and feedback mechanisms in cell signalling.
Here we established a new methodology for potential drug screening by combining measurement of the morphologic drug effect and evaluation of PET tracer uptake. The method could serve dual purposes: to evaluate effects of new drugs and to identify the optimal PET biomarker for early evaluation of drug effect.
Initially we investigated the nature of [18F]FDG uptake in MTS. We verified a reduced metabolism after either challenging with low temperature, or competition by unlabeled glucose or inhibition with Cytochalasin-B. We also observed a transport increase with insulin addition. These sets of tests confirmed that in MCF-7 MTSs, [18F]FDG accumulation is a biomarker which is related to transport via the specific glucose transporters GLUT, followed by phosphorylation by hexokinase.
An important aspect in the evaluation of chemotherapy by PET-FDG is whether [18F]FDG can be used as a biomarker to indicate treatment response. The accepted criterion for treatment response is reduction in tumour size or verified lack of further growth. This can in turn relate to a variety of functional aspects, such as induction of apoptosis, direct cell kill, effects on vascularity, reduced proliferation, remodelling of proportions of tumour and other cells etc. These aspects do not necessarily relate to effects on FDG uptake. E.g. transient increase in uptake of FDG has been postulated to be an indication of a positive effect causing a temporary inflammatory response, metabolic flare. A retardation of cellular proliferation can be associated with a lack of effect on FDG uptake. Hence it is of utmost importance to clarify the relation between antitumoral effects and effects on FDG uptake. For this, we believe that MTS are the best in vitro cellular model, allowing an easy evaluation of FDG uptake plus a means to observe effects on cellular growth. The method is ideally associated with evaluations of apoptosis induction, proliferation and other cellular biomarkers.
Paclitaxel and Docetaxel has a known activity against a broad range of tumour types, including breast, ovarian, lung, head and neck cancers [22
]. These potent anti-neoplastic drug binds to the N-terminal region of b-tubulin and promotes the formation of highly stable microtubules that resist depolymerization, thus preventing normal cell division and arresting the cell cycle at the G2-M phase [23
]. Combining [18
F]FDG-PET and image analysis, we monitored the effect of Paclitaxel for 3 days on two different human breast cancer line, in a concentration of 10 nM, 100 nM and 1 μM, added after 5 days of growth as MTS. In BT474, we observed reduced glucose transport per viable volume already at the lowest dose and the shorter treatment time. The effect was more moderate in MCF-7. These results indicate disturbance in cells physiology as a consequence of the treatment with taxanes that can be recorded with [18
Doxorubicin is an anthracycline antibiotic produced by the fungus streptomyces peucetius. It damages DNA by intercalation of the anthracycline portion, metal ion chelation, or by generation of free radicals. Doxorubicin has also been shown to inhibit DNA topoisomerase II which is critical to DNA function [24
]. Cytotoxic activity is not specific to cell cycle phase [27
]. Doxorubicin is also known to be activated by mitochondrial electron transport system and causes mitochondrial energy failure. Thus, the effect might be not only by DNA damage, but also by energy metabolism damage[28
]. In our experiments Doxorubicin decreased glucose transport at the highest dose. The data suggest that [18
F]FDG would record an antitumoral effect of Doxorubicin in the early phase of treatment at relevant doses. The effect of Doxorubicin was more intense on MCF-7 than on BT474. γH2AX formation that indicates DNA fragmentation [29
] was drastically increased in Doxorubicin treated BT474 MTSs. This radically alteration was not observed in FDG-uptake. It opens the discussion if [18
F]FDG is always the ideal biomarker for follow-up of all category of anticancer treatment.
Tamoxifen is a synthetic non-steroidal anti-oestrogen. It is thought to competitively block oestrogen receptors. Other biochemical effects of Tamoxifen include interaction with protein kinase C and stimulation of human NK cells [23
]. As expected, the effect of Tamoxifen can only be observed in MCF-7. In our experiments the effect of Tamoxifen can only be related to a glucose-transport alteration [31
Imatinib mesylate is a protein-tyrosine kinase inhibitor that inhibits the Bcr-Abl tyrosine kinase, the constitutive abnormal tyrosine kinase created by the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML)[32
]. It inhibits proliferation and induces apoptosis in Bcr-Abl positive cell lines as well as fresh leukemic cells from Philadelphia chromosome positive chronic myeloid leukemia [33
Imatinib is not entirely selective; it also inhibits the receptor tyrosine kinases for platelet-derived growth factor (PDGF), stem cell factor (SCF), c-Kit and thereby inhibits PDGF- and SCF-mediated cellular events [34
]. In our experiments no effect of Imatinib was observed.