In vitro and in vivo evaluation of the radiosensitizing effect of a selective FGFR inhibitor (JNJ-42756493) for rectal cancer.

BACKGROUND: We examined the anti-tumor effect and radiosensitizing potential of a small molecule inhibitor of fibroblast growth factor receptor (FGFR) in colorectal cancer (CRC) in vitro and in vivo. METHODS: Effects of in vitro drug treatment on cell survival, proliferation, FGFR signaling, cell cycle distribution, apoptosis and radiosensitivity were assessed using various CRC cell lines with FGFR wild type (Caco2 and HCA7) and FGFR2 amplification (HCT116, NCI-H716). In vivo tumor responses to FGFR inhibition with and without radiation therapy were evaluated by growth delay assays in two colorectal xenograft mouse models (NMRI nu/nu mice injected with NCI-H716 or CaCo2 cells). Mechanistic studies were conducted using Western blot analysis, immunohistochemistry and qPCR. RESULTS: In the tested cell lines, the FGFR inhibitor (JNJ-42756493) was effective in vitro and in vivo in CRC tumors with highest expression of FGFR2 (NCI-H716). In vitro, cell proliferation in this line was decreased, associated with increased apoptotic death and decreased cell survival. In vivo, growth of NCI-H716 tumors was delayed by 5 days by drug treatment alone, although when drug delivery was stopped the relative tumor volume increased compared to control. The FGFR inhibitor did not radiosensitize NCI-H716 tumors either in vitro or in vivo. CONCLUSIONS: Among tested CRC cell lines, the growth inhibitory activity of this FGFR inhibitor was evident in cell lines with high constitutive FGFR2 expression, suggesting that FGFR addiction may provide a window for therapeutic intervention, though caution is advised. Preclinical study with NCI-H716 and Caco2 tumor demonstrated that continued presence of drug could be essential for tumor growth control, especially in cells with aberrant FGFR expression. In the tested set-up, the inhibitor showed no radiosensitizing effect.

Investigation of possible endogenous hypoxia markers in colorectal cancer.

OBJECTIVE: We evaluated the potential of some recently proposed hypoxia markers, being monocarboxylic acid transporter 1 (MCT1), MCT4 and prolyl hydroxylase 2 (PHD2); and a more established hypoxia marker, glucose transporter-1 (GLUT-1), by testing the association with the exogenous marker pimonidazole. MATERIALS AND METHODS: Paraffin embedded tumour sections of 20 colorectal cancer patients were stained for blood vessels together with either pimonidazole or carbonic anhydrase-IX (CA-IX) and single stained for MCT1, MCT4, GLUT-1, and PHD2. Expression of all markers was compared with expression of pimonidazole and micro-vessel density (MVD) and with disease-free survival (DFS) and overall survival (OS). RESULTS: No correlation was found between the different intrinsic hypoxia markers tested and pimonidazole. A trend for high MCT1 expression in biopsies with low CA-IX expression was found (R = -0.45, p = 0.06) and also the expression of MCT1 was higher in tumours with a high MVD (R = 0.49, p = 0.04). The more advanced tumours showed a higher expression of GLUT-1 (p = 0.03). A low CA-IX expression in the tumour correlated with better DFS (p = 0.03) and related to better OS (p = 0.07). CONCLUSION: Although none of the tested intrinsic hypoxia markers correlated with pimonidazole staining, we confirmed the important role of both GLUT-1 and CA-IX for a more advanced pTNM (pathological tumour-node-metastasis) stage and DFS respectively.

Molecular imaging of therapy response with (18)F-FLT and (18)F-FDG following cyclophosphamide and mTOR inhibition.

PURPOSE: Evaluation and comparison of 3'-[(18)F]-fluoro-3'-deoxy-L-thymidine (FLT) and 2-[(18)F]-fluoro-2-deoxyglucose (FDG)-PET to monitor early response following both cyclophosphamide and temsirolimus treatment in a mouse model of Burkitt lymphoma. METHODS: Daudi xenograft mice were treated with either cyclophosphamide or temsirolimus and imaged with FLT-PET and FDG-PET on appropriate days post therapy inititiation. Immunohistochemical (IHC) studies (H&E, TUNEL, CD20, PCNA and ki-67) and DNA flow cytometry studies were performed. RESULTS: FDG tumor uptake decreased immediately after cyclophosphamide treatment while FLT-PET showed only a late and less pronounced decrease. A fast induction of apoptosis was observed together with an early accumulation of cells in the S-phase of the cell cycle, suggesting DNA repair. Temsirolimus treatment reduced both FDG and FLT tumor uptake immediately after therapy and resulted in a fast induction of apoptosis and G(0)-G(1) phase accumulation. CONCLUSION: FLT response was less distinct than FDG response and may be controlled by DNA repair early after cyclophosphamide. Nevertheless, FLT-PET was able to reflect decreased proliferation following temsirolimus.

Bioluminescence imaging of therapy response does not correlate with FDG-PET response in a mouse model of Burkitt lymphoma.

Since the development and evaluation of novel anti-cancer therapies require molecular insight in the disease state, both FDG-PET and BLI imaging were evaluated in a Burkitt B-cell lymphoma xenograft model treated with cyclophosphamide or temsirolimus. Daudi xenograft mice were treated with either cyclophosphamide or temsirolimus and imaged with BLI and FDG-PET on d0 (before treatment), d2, d4, d7, d9 and d14 following the start of therapy. Besides tumor volume changes, therapy response was assessed with immunohistochemical analysis (apoptosis). BLI revealed a flare following both therapeutics that was significantly higher when compared to control tumors. FDG-PET decreased immediatelly, long before the tumor reduced in size. Late after therapy, BLI signal intensities decreased significantly compared to baseline subsequent to tumor size reduction while apoptosis was immediately induced following both treatment regimen. Unlike FDG, BLI was not able to reflect reduced levels of viable cells and was not able to predict tumor size response and apoptosis response.

Radioiodinated phenylalkyl malonic acid derivatives as pH-sensitive SPECT tracers.

INTRODUCTION: In vivo pH imaging has been a field of interest for molecular imaging for many years. This is especially important for determining tumor acidity, an important driving force of tumor invasion and metastasis formation, but also in the process of apoptosis. METHODS: 2-(4-[(123)I]iodophenethyl)-2-methylmalonic acid (IPMM), 2-(4-[(123)I]iodophenethyl)-malonic acid (IPM), 2-(4-[(123)I]iodobenzyl)-malonic acid (IBMM) and 4-[(123)I]iodophthalic acid (IP) were radiolabeled via the Cu(+) isotopic nucleophilic exchange method. All tracers were tested in vitro in buffer systems to assess pH driven cell uptake. In vivo biodistribution of [(123)I]IPMM and [(123)I]IPM was determined in healthy mice and the pH targeting efficacy in vivo of [(123)I]IPM was evaluated in an anti-Fas monoclonal antibody (mAb) apoptosis model. In addition a mouse RIF-1 tumor model was explored in which tumor pH was decreased from 7.0 to 6.5 by means of induction of hyperglycemia in combination with administration of meta-iodobenzylguanidine. RESULTS: Radiosynthesis resulted in 15-20% for iodo-bromo exchange and 50-60% yield for iodo-iodo exchange while in vitro experiments showed a pH-sensitive uptake for all tracers. Shelf-life stability and in vivo stability was excellent for all tracers. [(123)I]IPMM and [(123)I]IPM showed a moderately fast predominantly biliary clearance while a high retention was observed in blood. The biodistribution profile of [(123)I]IPM was found to be most favorable in view of pH-specific imaging. [(123)I]IPM showed a clear pH-related uptake pattern in the RIF-1 tumor model. CONCLUSION: Iodine-123 labeled malonic acid derivates such as [(123)I]IPM show a clearly pH dependent uptake in tumor cells both in vitro and in vivo which allows to visualize regional acidosis. However, these compounds are not suitable for detection of apoptosis due to a poor acidosis effect.

Site-specific 68Ga-labeled Annexin A5 as a PET imaging agent for apoptosis.

PURPOSE: Two variants of Annexin A5 (Cys2-AnxA5 and Cys165-AnxA5) were labelled with Gallium-68 in order to evaluate their biological properties. PROCEDURES: Biodistribution and pharmacokinetics of the radiotracers were studied with µPET in healthy mice and in a mouse model of hepatic apoptosis. µPET imaging after IV injection of the tracers in combination with µMRI was performed in Daudi tumor bearing mice before and after treatment with a combination of chemotherapy and radiotherapy. RESULTS: The biodistribution data indicated a fast urinary clearance with only minor hepatobilliary clearance, although a high retention in the kidneys was observed. Animals treated with anti-Fas showed a 3 to 8 times higher liver uptake as compared to healthy animals. Tumor uptake of (68)Ga-Cys2-AnxA5 and (68)Ga-Cys165-AnxA5 was low but significantly increased after therapy. CONCLUSION: Both (68)Ga-Cys2-AnxA5 and (68)Ga-Cys165-AnxA5 show a clear binding to apoptotic cells and are promising tracers for rapid evaluation of cancer therapy.

Preclinical imaging of therapy response using metabolic and apoptosis molecular imaging.

PURPOSE: Early after therapy, 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) imaging is not always reliable due to the influx of inflammatory cells while apoptosis imaging offers a direct and early measurement of therapy effects. This study uses an improved apoptosis probe ((99m)Tc-hAnxA5) in combination with [(18)F]FDG imaging to evaluate therapy response. PROCEDURES: Daudi tumor tissue was implanted in the spleen of SCID mice. Treatment was performed with adriamycin and cyclophosphamide. Sequential [(18)F]FDG-positron emission tomography (PET) was acquired over 6 days and (99m)Tc-hAnxA5-SPECT was performed before and 1 day after therapy. RESULTS: On day 1, therapy induced apoptosis was visualized with (99m)Tc-hAnxA5 without a measurable change in [(18)F]FDG uptake. [(18)F]FDG uptake decreased significantly on day 3 and was even more pronounced on day 6. CONCLUSION: In this preclinical model, (99m)Tc-hAnxA5 imaging was able to detect apoptosis before metabolic changes were measured. These results confirm the value of apoptosis imaging for therapy response and give more insight in [(18)F]FDG imaging and its parameters to evaluate response.

Development and evaluation of a 68Ga labeled pamoic acid derivative for in vivo visualization of necrosis using positron emission tomography.

In this study, we labeled N,N'-bis(diethylenetriamine pentaacetic acid)-pamoic acid bis-hydrazide (bis-DTPA-PA) with the generator produced PET radionuclide gallium-68 and evaluated 68Ga-bis-DTPA-PA as a potential tracer for in vivo visualization of necrosis by positron emission tomography (PET). Radiolabeling was achieved with a decay-corrected radiochemical yield of 63%. Biodistribution and in vivo stability studies in normal mice showed that 68Ga-bis-DTPA-PA is cleared faster from normal tissue than the previously reported 99mTc(CO)3 complex with bis-DTPA-PA which on the other hand is more stable in vivo. 68Ga-bis-DTPA-PA showed a 3.5-5 times higher binding to necrotic tissue than to viable tissue as shown by in vitro autoradiography while no statistically significant increased hepatic uptake was found in a biodistribution study in a mouse model of hepatic apoptosis. Specificity and avidity for necrosis was further evaluated in rats with a reperfused partial liver infarction and ethanol induced muscular necrosis. Dynamic microPET images showed a fast and prolonged uptake of 68Ga-bis-DTPA-PA in necrotic tissue with in vivo and ex vivo images correlating well with histochemical stainings. With necrotic to viable tissue activity ratios of 8-15 on ex vivo autoradiography, depending on the necrosis model, 68Ga-bis-DTPA-PA showed a faster and higher uptake in necrotic tissue than the 99mTc(CO)3 analog. These results show that 68Ga-bis-DTPA-PA specifically binds to necrotic tissue and is a promising tracer for in vivo visualization of necrosis using PET.

18F-FLT and 18F-FDG PET to measure response to radiotherapy combined with celecoxib in two colorectal xenograft models.

PURPOSE: To determine the dependence of celecoxib on the tumour micro-environment in vitro and in vivo and to compare the use of (18)F-Fluorodeoxyglucose ((18)F-FDG) and (18)F- 3'-deoxy-3-fluorothymidine ((18)F-FLT) to measure tumour response. MATERIALS AND METHODS: In vitro, colony assays were performed on a cyclo-oxygenase 2 (COX-2) negative (HCT116) and a COX-2 positive cell line (HCA7). Xenograft models of these cell lines were treated with celecoxib and/or radiotherapy. Micro Positron Emission Tomography (microPET) scans with (18)F-FDG and (18)F-FLT were performed at different time-points. RESULTS: In vitro, no radiosensitising effect was seen in either of the cell lines. In vivo results showed a significant effect of celecoxib in the COX-2 negative tumours (HCT116) (enhancement ratio 1.5, p = 0.02) while no significant effect was observed in the COX-2 positive model (HCA7). A good correlation between (18)F-FDG and (18)F-FLT uptake was seen in both tumour models (r = 0.48, p = 0.002; r = 0.41, p = 0.005). After irradiation, a decrease in the uptake of both tracers was observed in both tumour models, which was more pronounced in the combination group, confirming the growth delay data. CONCLUSIONS: The contradicting in vitro and in vivo results suggest a major role of the tumour micro-environment. (18)F-FLT seems a good alternative for (18)F-FDG to follow tumour growth after radiation treatment.