Accumulation of Tc‐99m HL91 in Tumor Hypoxia: In Vitro Cell Culture and In Vivo Tumor Model

Abstract

Hypoxic cells within a tumor can account, in part, for resistance to radiotherapy and chemotherapy. Indeed, the oxygenation status has been shown to be a prognostic marker for the outcome of therapy. The purpose of this study was to determine whether Tc-99m HL91 (HL91), a noninvasive imaging tracer, detects tumor hypoxia in vitro in cell culture and in vivo in a tumor model. Uptake of HL91 in vitro into human lung cancer cells (A549) and murine Lewis lung cancer cells (LL2) was investigated at oxygen concentrations of 20% O2 (normoxia), and 1% O2 (hypoxia). HL91 biodistribution was studied in four groups: severe combined immune deficiency (SCID) mice bearing A549 tumors, C57BL/6NCrj (B6) mice bearing LL2 tumors, SCID controls, and B6 controls. Accumulation of the tracer was compared between tumors treated with hydralazine or phosphate-buffered saline (PBS). Scintigraphic images were obtained for hydralazine-treated mice and PBS-treated mice in each of the four study groups. Autoradiography of tumor slices was also acquired. In vitro studies identified hypoxia-selective uptake of HL91, with significantly increased uptake in the hypoxic state than in the normoxic state. Biodistribution and scintigraphy showed increased HL91 uptake during tumor hypoxia at 0.5 hours, and there was progressively increased activity for up to 4 hours after tracer administration. HL91 accumulation in tumor hypoxia was markedly increased in mice treated with hydralazine compared with those treated with PBS. Autoradiography revealed high HL91 uptake in the peripheral areas around the necrotic regions of the tumor, which were identified by histologic examination. HL91 exhibits selectivity for tumor hypoxia both in vitro and in vivo and provides a successful imaging modality for the detection of tumor hypoxia in vivo.

Hypoxia imaging predicts success of hypoxia-induced cytosine deaminase/5-fluorocytosine gene therapy in a murine lung tumor model

“…7 Our previous study demonstrated that the uptake of Tc-99m-HL91 in LL2 cells was increased by approximately 10-16-fold under hypoxic conditions compared with normoxic conditions throughout the 4-h study. 19 Moreover, the use of Tc-99m-HL91 has been described for identifying tumor hypoxia in vitro and ex vivo. 17,[19][20][21][22][23][24] The present study demonstrates for the first time, to our knowledge, that Tc99m-HL91 is useful as an imaging biomarker to predict the treatment responses of hypoxia-regulated CD/5-FC gene therapy in a mouse lung tumor model.…”

“…In our result of autoradiography, high uptake of Tc-99m-HL91 corresponded to the histopathologically hypoxic area, consistent with previous reports. [15][16][17][18][19][20][21][22][23][24] Tc-99m-HL91 displays increased retention in hypoxic tumors and is correlated with independent measures of tissue oxygen tension. 15 An oxygen-specific retention mechanism may determine the amount of the radiotracer that is trapped in hypoxic tissues.…”

“…18 In vitro studies have identified hypoxia-selective uptake of Tc-99m-HL91, with a significant increase in the uptake in the hypoxic state compared with the normoxic state. 17,19,20 Animal studies have also shown that Tc99m-HL91 is markedly increased in mice treated with hydralazine compared with controls. 19,20 The aim of this study was to test our hypothesis that Tc-99m-HL91 imaging can be used to identify the efficacy of hypoxia-induced CD gene/5-FC gene therapy for cancer.…”

“…17,19,20 Animal studies have also shown that Tc99m-HL91 is markedly increased in mice treated with hydralazine compared with controls. 19,20 The aim of this study was to test our hypothesis that Tc-99m-HL91 imaging can be used to identify the efficacy of hypoxia-induced CD gene/5-FC gene therapy for cancer. Our results show that the tumor uptake of Tc-99m-HL91 can predict the antitumor response of the CD/5-FC cancer gene therapy.…”

“…A continuous flow of warmed, humidified gas containing 20% O 2 , 5% CO 2 and 75% N 2 for normoxia or 1% O 2 , 5% CO 2 and 90% N 2 for hypoxia was passed over the cells. 19 The pO 2 was monitored by an oxygen analyzer that controlled an automated valve system (Pro-Ox, BioSpherix, Redfield, NY). After 48 h, the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 0.5 mg ml À1 final concentration; Sigma) assay was performed to determine cell viability.…”