Process to Remove the Size Variants Contained in the Antibody-Chelator Complex PCTA-NCAB001 for Radiolabeling with Copper-64.

Understanding the physicochemical properties of antibody-drug conjugates is critical to assess their quality at manufacturing and monitor them during subsequent storage. For radiometal-antibody complexes, it is important to control the properties of the antibody-chelator conjugate to maintain the quality of the final product. We have been developing 64Cu-labeled anti-epidermal growth factor receptor antibody NCAB001 (64Cu-NCAB001) for the early diagnosis and therapy of pancreatic cancer with positron-emission tomography. Here, we characterized the larger size variants contained in the antibody-chelator conjugate PCTA-NCAB001 by multi-angle light scattering coupled with size-exclusion chromatography. Secondly, we developed a chromatographic method to remove these size variants. Lastly, we demonstrated the stability of PCTA-NCAB001 after the removal of size variants. Dimer and oligomers were identified in PCTA-NCAB001. These larger size variants, together with some smaller size variants, could be removed by hydrophobic interaction chromatography. The PCTA-NCAB001 product, after the removal of these size variants, could be stored at 4 °C for six months. The methods developed here can be applied to assure the quality of PCTA-NCAB001 and other antibody-drug conjugates to facilitate the development of antibody-radiometal conjugates for positron-emission tomography and radioimmunotherapy of malignant cancers.

Preclinical Safety Evaluation of Intraperitoneally Administered Cu-Conjugated Anti-EGFR Antibody NCAB001 for the Early Diagnosis of Pancreatic Cancer Using PET.

Detecting tumor lesions <1 cm in size using current imaging methods remains a clinical challenge, especially in pancreatic cancer. Previously, we developed a method to identify pancreatic tumor lesions ≥3 mm using positron emission tomography (PET) with an intraperitoneally administered 64Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody (64Cu-NCAB001 ipPET). Here, we conducted an extended single-dose toxicity study of 64Cu-NCAB001 ipPET in mice based on approach 1 of the current ICH M3 [R2] guideline, as our new drug formulation contains 45 µg of the antibody. We used NCAB001 labeled with stable copper isotope instead of 64Cu. The total content of size variants was approximately 6.0% throughout the study. The relative binding potency of Cu-NCAB001 to recombinant human EGFR was comparable to that of cetuximab. The general and neurological toxicities of Cu-NCAB001 ipPET at 62.5 or 625 µg/kg were assessed in mice. The no-observed-adverse-effect level of Cu-NCAB001 was 625 µg/kg, a dose approximately 1000-fold higher at the µg/kg level than the dose of 64Cu-NCAB001 in our formulation (45 µg). The size variants did not affect the safety of the formulation. Therefore, clinical studies on the efficacy of 64Cu-NCAB001 ipPET for early detection of pancreatic cancer using PET imaging can be safely conducted.

Evaluation of 64Cu-Labeled New Anti-EGFR Antibody NCAB001 with Intraperitoneal Injection for Early PET Diagnosis of Pancreatic Cancer in Orthotopic Tumor-Xenografted Mice and Nonhuman Primates.

OBJECTIVES: To improve the prognosis of pancreatic cancer, new imaging methods to identify tumor lesions at a size of <1 cm are urgently needed. To approach this clinical issue, we developed a new method to detect small tumor lesions in the pancreas (≥3 mm) by positron emission tomography (PET) using an intraperitoneally (ip)-administered 64Cu-labeled new anti-epidermal growth factor receptor (EGFR) antibody (encoded as NCAB001), called 64Cu-NCAB001 ipPET. METHODS: NCAB001 was manufactured under cGMP conditions and labeled with 64Cu. The radiochemical and biological properties of 64Cu-NCAB001 were evaluated. Tumor uptake of an ip-administered 64Cu-NCAB001 in mice with orthotopic pancreatic tumor xPA1-DC xenografts was also evaluated. Pharmacokinetics and radiation dosimetry were examined using PET images acquired after the ip administration of 64Cu-NCAB001 into cynomolgus monkeys with pharmacologic safety monitoring. RESULTS: Radio-chromatography, cell-binding assays, and biodistribution of 64Cu-NCAB001 in mice were identical to those of our previous data with clinically available cetuximab. Small tumor lesions in the pancreas (≥3 mm) of mice could be identified by 64Cu-NCAB001 ipPET. The ip administration of 64Cu-NCAB001 into monkeys was safely conducted using ultrasound imaging. PET images in monkeys showed that ip-administered 64Cu-NCAB001 was distributed throughout the intraperitoneal cavity for up to 6 h and cleared thereafter. Most of the radioactivity was distributed in the liver and the large intestine. The radioactivity around the pancreas became negligible 24 h after administration. The estimated human effective dose was 0.0174 mSv/MBq. CONCLUSION: Our data support the initiation of clinical trials of 64Cu-NCAB001 ipPET to transfer this promising tool for the early diagnosis of pancreatic cancers.

Clinical practice guidelines for high-resolution breast PET, 2019 edition.

Breast positron emission tomography (PET) has had insurance coverage when performed with conventional whole-body PET in Japan since 2013. Together with whole-body PET, accurate examination of breast cancer and diagnosis of metastatic disease are possible, and are expected to contribute significantly to its treatment planning. To facilitate a safer, smoother, and more appropriate examination, the Japanese Society of Nuclear Medicine published the first edition of practice guidelines for high-resolution breast PET in 2013. Subsequently, new types of breast PET have been developed and their clinical usefulness clarified. Therefore, the guidelines for breast PET were revised in 2019. This article updates readers as to what is new in the second edition. This edition supports two different types of breast PET depending on the placement of the detector: the opposite-type (positron emission mammography; PEM) and the ring-shaped type (dedicated breast PET; dbPET), providing an overview of these scanners and appropriate imaging methods, their clinical applications, and future prospects. The name "dedicated breast PET" from the first edition is widely used to refer to ring-shaped type breast PET. In this edition, "breast PET" has been defined as a term that refers to both opposite- and ring-shaped devices. Up-to-date breast PET practice guidelines would help provide useful information for evidence-based breast imaging.

Characterization and Stabilization of a New 64Cu-Labeled Anti-EGFR Antibody NCAB001 for the Early Detection of Pancreatic Cancer with Positron Emission Tomography.

Early diagnosis of pancreatic cancer using current imaging modalities remains challenging. We have developed a new approach to identify tumor lesions ≥ 3 mm in the pancreas by positron emission tomography (PET) with a new intraperitoneally administered 64Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody (encoded as NCAB001), called 64Cu-NCAB001 ipPET. Generally, in clinical research, a radiometal-antibody complex must be prepared immediately before use at the imaging site. To make 64Cu-NCAB001 ipPET available to daily clinical practices in a sustainable way, the NCAB001-chelator conjugate and 64Cu-NCAB001 must be characterized and stabilized. NCAB001 was manufactured under cGMP conditions. NCAB001 was conjugated with a bifunctional chelator (p-SCN-Bn-PCTA), and the antibody-chelator conjugate (PCTA-NCAB001) was characterized by LC/MS and ELISA. Thereafter, to effectively manufacture 64Cu-NCAB001, we developed a new formulation to stabilize PCTA-NCAB001 and 64Cu-NCAB001. An average of three PCTA chelators were conjugated per molecule of NCAB001. The relative binding potency of PCTA-NCAB001 was comparable to cetuximab. The formulation consisting of acetate buffer, glycine, and polysorbate-80 stabilized PCTA-NCAB001 for a year-long storage. Additionally, this formulation enabled the stabilization of 64Cu-NCAB001 for up to 24 h after radiolabeling with a sufficient radioactivity concentration for clinical use. These results may accelerate the future use of 64Cu-NCAB001 ipPET in clinical settings for the early diagnosis and treatment of pancreatic cancer.

Development of an Isolator System for PET Drug Compounding with Sterilization and Dispensing Units.

To maintain sterility of PET drug is the most important for in-house positron emission tomography (PET) drug manufacturing, and sanitary control of the laboratory to perform aseptic procedure is the key point for the sterility of PET drugs. However, rigorous sanitary control affects both the high cost and the low efficiency. To conquer those, we developed an isolator system especially for PET drug compounding including sterilization and dispensing units. This system consists of a HEPA unit for inlet and outlet, positive regulation of the ear inside isolator, a sterilizer with vapored hydrogen peroxide and a dispenser with self-shield for radiation. We set the materials for the dispenser through gloves, and the compounding such as sterilization and dispensing PET drugs to the containers is performed automatically without radiation. High level assurance of PET drug sterility is expected to be accomplished in the PET centers of the hospitals without high level sanitary control.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells.

[Assessment of validity of the archived standard curve in endotoxin assay, produced in other facilities].

We have reported the possibility of the use of the archived standard curve of endotoxin assay, which is prepared in the same facility from the viewpoint of the accuracy and precision. In this study, the possibility of the use of the archived standard curves prepared in the different facilities was investigated with the same data set in the previous paper. The evaluation was performed with the recovery rate of the concentrations of the standard solutions, as the same method as the previous study. The clotting times of the standard solutions were substituted into the standard curves prepared in the different facilities from those, in which standard solutions were prepared. The recovery rates were 86.1-125.0%, and the range was almost the same as that when the facility preparing standard solutions were the same as that preparing the standard curve. From this data, if the protocols of the preparation of standard solutions, such as mixing and the interval timing until set to the apparatus and so on, can be set the same between the endotoxin test and the preparation of the archived standard curves, the endotoxin concentration calculated with the archived standard curves prepared in other facilities were not varied very much, compared to the true values and the values obtained from the use of the archived standard curves prepared in the same facility.

Fatty acid synthase is a key target in multiple essential tumor functions of prostate cancer: uptake of radiolabeled acetate as a predictor of the targeted therapy outcome.

Fatty acid synthase (FASN) expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In addition, how FASN inhibition affects tumor progression remains unclear. Here, we showed the method to predict FASN-targeted therapy outcome using radiolabeled acetate uptake and presented mechanisms of FASN inhibition with human prostate cancer cell lines, to provide the treatment strategy of FASN-targeted therapy. We revealed that tumor uptake of radiolabeled acetate reflected the FASN expression levels and sensitivity to FASN-targeted therapy with orlistat in vitro and in vivo. FASN-targeted therapy was noticeably effective against tumors with high FASN expression, which was indicated by high acetate uptake. To examine mechanisms, we established FASN knockdown prostate cancer cells by transduction of short-hairpin RNA against FASN and investigated the characteristics by analyses on morphology and cell behavior and microarray-based gene expression profiling. FASN inhibition not only suppressed cell proliferation but prevented pseudopodia formation and suppressed cell adhesion, migration, and invasion. FASN inhibition also suppressed genes involved in production of intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression. Collectively, our data demonstrated that uptake of radiolabeled acetate is a useful predictor of FASN-targeted therapy outcome. This suggests that [1-(11)C]acetate positron emission tomography (PET) could be a powerful tool to accomplish personalized FASN-targeted therapy by non-invasive visualization of tumor acetate uptake and selection of responsive tumors. FASN-targeted therapy could be an effective treatment to suppress multiple steps related to tumor progression in prostate cancers selected by [1-(11)C]acetate PET.

[Assessment of validity of the archived standard curve in endotoxin assay].

The archived standard curve of endotoxin assay was evaluated to be possible to be used for the endotoxin assay as the reliable standard curve, instead the standard curve was produced each time of the assay. The archived standard curve shall be produced from three standard curves for three days, following the guidance issued from FDA in 1991, and the evaluation whether the archived standard curves can be applicable to use daily was performed with the recovery rate of the concentrations obtained from the archived standard curves against the true values. The three case studies were prepared: (1) the same person, who prepared the archived standard curves, performed this assay with the standard solutions (repeatability condition with the same tester, at the same facility), (2) the person, who did not prepare the archived standard curves, performed this assay with standard solutions (reproducibility condition with the different tester and dates), (3) the same preparation as (1), but using different three lots of lysates. The recovery rates were (1) 85-127%, (2) 86-124%, (3) 64-156%, respectively. From this data, the endotoxin concentration calculated with the archived standard curves were not varied very much, compared to the true values, but further discussion are necessary when the archived standard curves would be applied in daily analysis of PET drugs, regarding the protocol, the requirement to use the archived standard curve and the daily internal control as system suitability tests.

The use of nanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cell migration and well-regulated spheroid formation.

Two-dimensional (2D) cell cultures are essential for drug development and tumor research. However, the limitations of 2D cultures are widely recognized, and a better technique is needed. Recent studies have indicated that a strong physical contact between cells and 2D substrates induces cellular characteristics that differ from those of tumors growing in vivo. 3D cell cultures using various substrates are then developing; nevertheless, conventional approaches have failed in maintenance of cellular proliferation and viability, uniformity, reproducibility, and/or simplicity of these assays. Here, we developed a 3D culture system with inorganic nanoscale scaffolding using nanoimprinting technology (nano-culture plates), which reproduced the characteristics of tumor cells growing in vivo. Diminished cell-to-substrate physical contact facilitated spontaneous tumor cell migration, intercellular adhesion, and multi-cellular 3D-spheroid formation while maintaining cellular proliferation and viability. The resulting multi-cellular spheroids formed hypoxic core regions similar to tumors growing in vivo. This technology allows creating uniform and highly-reproducible 3D cultures, which is easily applicable for microscopic and spectrophotometric assays, which can be used for high-throughput/high-content screening of anticancer drugs and should accelerate discovery of more effective anticancer therapies.

Copper-64-diacetyl-bis (N4-methylthiosemicarbazone) accumulates in rich regions of CD133+ highly tumorigenic cells in mouse colon carcinoma.

INTRODUCTION: (64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a potential imaging agent of hypoxic tumor for use with PET. Recent literature demonstrated that cancer cells expressing CD133, which is a frequently used marker for so-called cancer stem cells or cancer stem cell-like cells (collectively referred to here as CSCs), contribute to tumor's therapeutic resistance and metastasis ability. Culturing under hypoxia is also reported to enlarge the proportion of CD133(+) cells, which would indicate survival advantage of CD133(+) cells under hypoxia. Here, we investigated the relationships between (64)Cu-ATSM accumulation and existence of CD133(+) cells using mouse colon carcinoma (colon-26) tumor. METHODS: Intratumor distribution of (64)Cu-ATSM and (18)F-fluorodeoxyglucose ((18)FDG) was compared with immunohistochemical staining for CD133 with a colon-26 model. In vitro characterization of CD133(+) colon-26 cells was also performed. RESULTS: In colon-26 tumors, (64)Cu-ATSM localized preferentially in regions with a high density of CD133(+) cells. The percentage of CD133(+) cells was 11-fold higher in (64)Cu-ATSM high-uptake regions compared with (18)FDG high- (but (64)Cu-ATSM low-) uptake regions. CD133(+) colon-26 cells showed characteristics previously linked with CSCs in other cancer cell lines, such as high colony-forming ability, high tumor-initiating ability and enrichment under hypoxic cultivation. The proportion of CD133(+) cells was enlarged by culturing under glucose starvation as well as hypoxia, and (64)Cu-ATSM uptake was increased under such conditions. CONCLUSIONS: Our findings showed that, in colon-26 tumors, (64)Cu-ATSM accumulates in rich regions of CD133(+) cells with characteristics of CSCs. Therefore (64)Cu-ATSM could be a potential imaging agent for rich regions of CD133(+) cells, associated with CSCs, within tumors.

Effect of multimerization of a linear Arg-Gly-Asp peptide on integrin binding affinity and specificity.

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study, mono-, di- and trimeric Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Tyr (AVTGRGDSY) peptides, labeled with (125)I or Cy5.5, were compared in vitro and in vivo. Using human embryonic kidney HEK293 (naturally alpha(V)-positive and beta(3)-negative), HEK293(beta(1)) (beta(1)-transfected and alpha(V)beta(3)-negative), HEK293(beta(3)) (beta(3)-transfected and strongly alpha(V)beta(3)-positive), and human glioblastoma U87MG (naturally alpha(V)beta(3)-positive) cell lines we evaluated their binding affinity and specificity. In vitro, the monomeric AVTGRGDSY showed specific binding to both HEK293(beta(1)) and HEK293(beta(3)) cells. Multimerization resulted in no change toward HEK293 cells, diminished binding with HEK293(beta(1)) cells, but substantially enhanced binding with alpha(V)beta(3)-positive HEK293(beta(3)) and U87MG cells. Moreover, multimeric AVTGRGDSY peptides were found to be nearly comparable to the same molar concentration of a well-known alpha(V)beta(3)-specific cyclo(RGDfV) (c(RGDfV)) peptide in specificity and affinity for targeting alpha(V)beta(3) integrin. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric analogue, the Cy5.5-labeled dimeric AVTGRGDSY peptide produced markedly enhanced tumor-to-background contrast in HEK293(beta(3)) tumor-bearing mice than in HEK293(beta(1)) tumor-bearing mice. In conclusion, the present study showed the difference of monomeric and multimeric linear Arg-Gly-Asp (RGD)-containing compound in integrin selectivity and affinity. Our data provide useful information for the design of novel RGD peptides.

Tumor uptake of radiolabeled acetate reflects the expression of cytosolic acetyl-CoA synthetase: implications for the mechanism of acetate PET.

INTRODUCTION: [1-(11)C]Acetate positron emission tomography (PET) is used for myocardial studies. In the myocardium, mitochondrial acetyl-CoA synthetase (ACSS1) mainly contributes to the radiopharmaceutical uptake. [1-(11)C]Acetate PET is also used for tumor diagnosis; however, the uptake mechanism of radiolabeled acetate in tumors remains unclear. Our previous study reported that cytosolic acetyl-CoA synthetase (ACSS2) was expressed in tumor cells and up-regulated under hypoxia, whereas expression of ACSS1 was negligible regardless of the oxygen conditions. We also indicated that ACSS2 is a bidirectional enzyme that controls acetyl-CoA/acetate metabolism in tumor cells. In this study, to elucidate the basic mechanism of tumor acetate uptake, we focused on ACSS2 and investigated the role of ACSS2 in the uptake of radiolabeled acetate in tumor cells. METHODS: [1-(14)C]Acetate uptake and ACSS2 expression were examined in four tumor cell lines under normoxia or hypoxia. An ACSS2 knockdown study was also performed. RESULTS: [1-(14)C]Acetate uptake was increased in the tumor cells under hypoxia. This pattern followed that of ACSS2 expression. The incorporated (14)C was mostly distributed in the lipid-soluble fractions, and this tendency increased under hypoxia. ACSS2 knockdown led to a corresponding reduction in [1-(14)C]acetate uptake in all tumor cell lines examined under normoxia and hypoxia. CONCLUSIONS: ACSS2 plays an important role in the uptake of radiolabeled acetate in tumor cells, which is different from that in the myocardium, which mainly involves ACSS1. The uptake of radiolabeled acetate in tumors increased under hypoxia along with up-regulation of ACSS2 expression. This suggests a possible mechanism for acetate PET for tumors.

Cytosolic acetyl-CoA synthetase affected tumor cell survival under hypoxia: the possible function in tumor acetyl-CoA/acetate metabolism.

Understanding tumor-specific metabolism under hypoxia is important to find novel targets for antitumor drug design. Here we found that tumor cells expressed higher levels of cytosolic acetyl-CoA synthetase (ACSS2) under hypoxia than normoxia. Knockdown of ACSS2 by RNA interference (RNAi) in tumor cells enhanced tumor cell death under long-term hypoxia in vitro. Our data also demonstrated that the ACSS2 suppression slowed tumor growth in vivo. These findings showed that ACSS2 plays a significant role in tumor cell survival under hypoxia and that ACSS2 would be a potential target for tumor treatment. Furthermore, we found that tumor cells excreted acetate and the quantity increased under hypoxia: the pattern of acetate excretion followed the expression pattern of ACSS2. Additionally, the ACSS2 knockdown led to a corresponding reduction in the acetate excretion in tumor cells. These results mean that ACSS2 can conduct the reverse reaction from acetyl-CoA to acetate in tumor cells, which indicates that ACSS2 is a bi-directional enzyme in tumor cells and that ACSS2 might play a buffering role in tumor acetyl-CoA/acetate metabolism.

Radiolabeled choline as a proliferation marker: comparison with radiolabeled acetate.

[11C]Choline is a potential tracer to detect tumors, especially brain and prostate cancers. The metabolism of [11C]choline defines the accumulation pattern of [11C]choline in tumors depicted by positron emission tomography. Choline is a precursor of phosphatidylcholine that is a major constituent of membrane lipids. Membrane lipid synthesis as well as DNA synthesis is activated during cell proliferation. We investigated the relation between [14C]choline metabolism and proliferative activity using 10 tumor cell lines and fibroblasts. [14C]Choline uptake was higher in tumor cells than in fibroblasts and was correlated with the proliferative activity, though the sensitivity of [14C]choline uptake to proliferative activity was less than that of [1-14C]acetate. [14C]Phosphocholine produced from [14C]choline by phosphorylation mainly contributed to this accumulation. [11C]Choline can be used for the evaluation of tumor proliferation through estimating choline kinase activity.

Evaluation of [76Br]FBAU 3',5'-dibenzoate as a lipophilic prodrug for brain imaging.

[76Br]FBAU is a potential PET tracer for assessing proliferation. This study proposes that [76Br]FBAU 3',5'-dibenzoate has higher blood-brain-barrier permeability than [76Br]FBAU itself and thus might be better suited for applications in the brain. The brain uptake indexes of the two compounds measured after carotid injection (29.6 +/- 13.9 for [76Br]FBAU 3',5'-dibenzoate, versus 10.0 +/- 8.7 for [76Br]FBAU) support this claim. Biodistribution study also showed that the brain accumulation of activity was higher in rats injected with [76Br]FBAU 3',5'-dibenzoate than with [76Br]FBAU (0.119+/-0.023 DUR at 1 h, versus 0.061 +/- 0.006). [76Br]FBAU 3',5'-dibenzoate was relatively stable in rat plasma, gradually being hydrolyzed to [76Br]FBAU exponentially with a calculated half-life of 0.8 h. DNA incorporation of [76Br]FBAU was also confirmed. The results presented support the hypothesis that the 3',5'-dibenzoate can act as a prodrug for FBAU and deliver more radiolabeled nucleoside to the brain.

Basis of FLT as a cell proliferation marker: comparative uptake studies with [3H]thymidine and [3H]arabinothymidine, and cell-analysis in 22 asynchronously growing tumor cell lines.

The usefulness of radiolabeled 3'-fluoro-3'-deoxythymidine (FLT), a thymidine derivative with affinity to cytoplasmic thymidine kinase 1 (TK1), as a tumor proliferation marker was evaluated using [3H]FLT and 22 cultured tumor cell lines. Asynchronously growing tumor cells were used for studies to mimic in vivo status of tumors. FLT uptake in each cell line was compared with [3H]thymidine ([3H]Thd) uptake and %S-phase fraction, both known as acceptable markers of proliferation. Uptake of the mitochondrial TK2 specific substrate [3H]arabinothymidine ([3H]AraT) was studied as a reference. Metabolic fate of FLT in tumor cells was also analyzed to elucidate the retention mechanism of FLT. [3H]FLT uptake was mildly correlated with the %S-phase fraction (r=0.76, p<0.0001) and correlated better with [3H]Thd uptake (r=0.88, p<0.0001). In contrast, the TK(2) specific substrate, [3H]AraT, was not significantly correlated with the %S-phase fraction (r=0.19, p=0.39), although it showed some correlation with the [3H]Thd uptake (r=0.47, p<0.05). Over 90% of radioactivity of [3H]Thd was found in the DNA fraction after 60 minutes incubation. In contrast, most of the radioactivity of [3H]FLT was found in the acid-soluble fraction (95%). [3H]FLT incorporation into the DNA fraction was negligible (0.2%). The [3H]AraT was mainly distributed in the acid-soluble fraction (70%) and the DNA fraction (20%). From our results, we concluded that FLT uptake in tumor cells reflects tumor cell proliferation. However, much more convincing validation is needed to clarify the difference between FLT and true substrates for DNA synthesis, like thymidine.