Assessment of regional tumor hypoxia using 18F-fluoromisonidazole and 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) positron emission tomography: Comparative study featuring microPET imaging, Po2 probe measurement, autoradiography, and fluorescent microscopy in the R3327-AT and FaDu rat tumor models

doi:10.1016/j.ijrobp.2004.12.057

International Journal of Radiation OncologyBiologyPhysics

Volume 61, Issue 5, 1 April 2005, Pages 1493-1502

https://doi.org/10.1016/j.ijrobp.2004.12.057 Get rights and content

Purpose: To compare two potential positron emission tomography (PET) tracers of tumor hypoxia in an animal model.

Methods and Materials: The purported hypoxia imaging agents ¹⁸F-fluoromisonidazole (FMISO) and ⁶⁴Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) were compared by serial microPET imaging of Fisher-Copenhagen rats bearing the R3327-AT anaplastic rat prostate tumor. Probe measurements of intratumoral Po₂ were compared with the image data. At the microscopic level, the relationship between the spatial distributions of ⁶⁴Cu (assessed by digital autoradiography) and tumor hypoxia (assessed by immunofluorescent detection of pimonidazole) was examined. ¹⁸F-FMISO and ⁶⁴Cu-ATSM microPET images were also acquired in nude rats bearing xenografts derived from the human squamous cell carcinoma cell line, FaDu.

Results: In R3327-AT tumors, the intratumoral distribution of ¹⁸F-FMISO remained relatively constant 1–4 h after injection. However, that of ⁶⁴Cu-ATSM displayed a significant temporal evolution for 0.5–20 h after injection in most tumors. In general, only when ⁶⁴Cu-ATSM was imaged at later times (16–20 h after injection) did it correspond to the distribution of ¹⁸F-FMISO. Oxygen probe measurements were broadly consistent with ¹⁸F-FMISO and late ⁶⁴Cu-ATSM images but not with early ⁶⁴Cu-ATSM images. At the microscopic level, a negative correlation was found between tumor hypoxia and ⁶⁴Cu distribution when assessed at early times and a positive correlation when assessed at later times. For the FaDu tumor model, the early and late ⁶⁴Cu-ATSM microPET images were similar and were in general concordance with the ¹⁸F-FMISO scans.

Conclusion: The difference in behavior between the R3327-AT and FaDu tumor models suggests a tumor-specific dependence of Cu-ATSM uptake and retention under hypoxic conditions.

Introduction

Regions of local hypoxia are a common feature of many human cancers, as evidenced by histologic studies, Po₂ probe measurements, and scintigraphic imaging (1, 2, 3, 4, 5). Moreover, tumor hypoxia is emerging as an important determinant of relapse-free survival and overall clinical outcome (6, 7, 8). This appears to be largely independent of the treatment modality used and implies that hypoxia is associated with a more aggressive tumor phenotype. There appear to be at least three distinct manifestations of the hypoxic phenotype:

1
Hypoxic cells are three times more resistant than aerobic cells to ionizing radiation of low linear energy transfer. These radiation types include the high-energy photons and electrons most commonly used in cancer therapy. It has been hypothesized that the failure to control localized cancer at normal tissue tolerance doses may be a result of radioresistance of hypoxic tumor cells (9).
2
The hypoxic environment is selective for genetically unstable tumor cells, and hypoxia is associated with tumors that are more likely to metastasize (6, 10, 11).
3
Hypoxia regulates the expression of genes that are primarily involved in oxygen supply and use through the hypoxia-inducible factor pathway (12). Overexpression of a number of these genes is associated with tumor progression or poor prognosis (13, 14, 15, 16).

Given the importance of hypoxia in tumor progression and the response to treatment, it is of great importance to develop noninvasive methods that can identify tumors with substantial hypoxic fractions. Knowledge of the overall hypoxic status of individual tumors and, by inference, the likelihood of distant metastases will influence the design of treatment strategies and may improve overall survival. In addition, knowledge of variations in the spatial distribution of hypoxia within tumors may be of value in the identification of local “hotspots” of resistance, genetic instability, or tumor aggressiveness.

Positron emission tomography (PET) holds promise for identifying tumor hypoxia at both global and local levels. A number of potential hypoxia-targeting molecules are now available that can be labeled with positron-emitting radionuclides. These include fluoromisonidazole (FMISO) (17), fluoro-erythronitroimidazole (18), and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide (19), all of which can be labeled with ¹⁸F; iodoazomycin arabinoside (20) and iodoazomycin galactopyranoside (21, 22), both of which can be labeled with ¹²⁴I; and Cu(II)-diacetyl-bis(N4- methylthiosemicarbazone (Cu-ATSM) (23, 24), which can be labeled with ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, or ⁶⁴Cu. Some of these agents have been the subject of clinical studies (17, 25).

The aim of the current study was to compare directly the time-dependent intratumoral distribution and hypoxic cell targeting of two promising hypoxic-cell PET radiotracers, ¹⁸F-FMISO and ⁶⁴Cu-ATSM. This was investigated by sequential injection of the two radiolabeled compounds into the same tumor-bearing rats, followed by serial microPET imaging. Additional comparative studies on the distribution of hypoxia within the animal tumors were performed by direct oxygen probe measurement and by immunohistochemical detection of pimonidazole in tumor sections.

Section snippets

Tumor model and experimental design

The animals were maintained and used according to the guidelines of the Memorial Sloan-Kettering Cancer Center (New York, NY). The Institutional Animal Care and Use Committee approved the experimental protocol.

The principal tumor model used was the syngeneic Dunning R3327-AT anaplastic prostate adenocarcinoma (26) growing in Fisher-Copenhagen rats. Approximately 2 × 10⁶ tumor cells in a single-cell suspension in 20 μL of phosphate-buffered saline was injected subcutaneously into the right hind

PET image analysis

Visual examination of the entire R3327-AT data set indicated certain common features among the images. For any particular animal, the ¹⁸F-FMISO images acquired at different times between 30 min and 4 h after injection were similar in 7 of 7 cases (i.e., no major changes occurred with time in the pattern of activity). Although a general tendency was noted for the distributions to become more focal at later times, no regions of high uptake were seen in the later images that were not also present

Discussion

To our knowledge, the present report represents the first systematic study of the comparative intratumoral distribution of hypoxia-targeting agents at multiple times after injection. The data showed that the uptake of ¹⁸F-FMISO and ⁶⁴Cu-ATSM within R3327-AT syngeneic and FaDu xenograft tumors growing in, respectively, Fisher-Copenhagen and nude rats was heterogeneous. The intratumoral distribution of ¹⁸F-FMISO was similar for imaging times between 30 min and 4 h after injection in both tumor

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Citation Excerpt :
Therefore it is not unexpected that our measured threshold values of pO2 for [64Cu]CuATSM, and indeed all the other tracers, are lower and probably more realistic than many reported in literature that were not directly measured. Pimonidazole immunohistochemistry is often used to characterise tissue hypoxia from an immunohistochemical perspective and as a “gold–standard” hypoxia marker against which to validate PET hypoxia tracer accumulation [27–31]. The binding characteristic of pimonidazole has been described by some as almost binary, showing strong binding at ≤10 mmHg O2 but none above 10 mmHg [32].
The pO₂ threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To identify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([¹⁸F]FMISO, [⁶⁴Cu]CuATSM, and analogues [⁶⁴Cu]CuATS, [⁶⁴Cu]CuATSE, [⁶⁴Cu]CuCTS, [⁶⁴Cu]CuDTS, [⁶⁴Cu]CuDTSE, [⁶⁴Cu]CuDTSM) with levels of radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia.
A custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic conditions with constant measurement of pO₂ in media using an OxyLab pO₂™ probe. HCT116 human colonic carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting, tracer uptake by gamma counting and pimonidazole binding by flow cytometry.
Radioresistance, pimonidazole binding and HIF1α expression increased gradually as pO₂ decreased between 25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO₂ levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells.
This study highlights an important mismatch in pO₂ thresholds between these PET tracers and other markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced radioresistance, pimonidazole uptake and HIF1α expression. Although their pO₂ thresholds do not match the threshold for resistance to conventionally fractionated radiotherapy (pO₂ 2.5–10 mmHg), their specificity for extreme hypoxia (pO₂ ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in stereotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.
Imaging Hypoxia
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Hypoxia is a well-established pathophysiologic feature of solid tumors that has demonstrated links to poor prognosis. It is a direct cause of radioresistance and also contributes to chemoresistance. In addition, it is immunosuppressive. Given these pleiotropic effects, there is strong rationale for developing strategies to image hypoxia. This chapter reviews the characteristics of hypoxia that enable it to be imaged. A critical review of the three major methods for imaging hypoxia (Optics, PET/SPECT, and Magnetic Resonance Imaging) is presented; the strengths and weaknesses of each method for preclinical and clinical uses are described.
Imaging of Tumor Hypoxia for Radiotherapy: Current Status and Future Directions
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Citation Excerpt :
The exact mechanism of retention of Cu-ATSM is not fully understood, but hypoxia selectivity is believed to involve a conversion of Cu2+ to Cu1+ at low oxygen conditions, which is trapped within cells. Whereas initial testing was encouraging,111 later studies revealed clear discordance between 64Cu-ATSM and other hypoxia markers (eg, pimonidazole) in some animal tumor models.112–114 In addition, it was shown that the time interval required for development of hypoxia selectivity varied widely112 and that 64Cu-ATSM was insensitive to treatments that alters tumor oxygenation.115
Tumor regions that are transiently or chronically undersupplied with oxygen (hypoxia) and nutrients, and enriched with acidic waste products, are common due to an abnormal and inefficient tumor vasculature, and a deviant highly glycolytic energy metabolism. There is compelling evidence that tumor hypoxia is strongly linked to poor prognosis since oxygen-deprived cells are highly resistant to therapy including radio- and chemotherapy, and survival of such cells is a primary cause of disease relapse. Despite a general improvement in cancer survival rates, hypoxia remains a formidable challenge. Recent progress in radiation delivery systems with improved spatial accuracy that allows dose escalation to hypoxic tumors or even tumor subvolumes, and the development of hypoxia-selective drugs, including bioreductive prodrugs, holds great promise for overcoming this obstacle. However, apart from one notable exception, translation of promising preclinical therapies to the clinic have largely been disappointing. A major obstacle in clinical trials on hypoxia-targeting strategies has been the lack of reliable information on tumor hypoxia, which is crucial for patient stratification into groups of those that are likely to benefit from intervention and those who are not. Further, in many newer trials on hypoxia-selective drugs the choice of cancer disease and combination therapy has not always been ideal, especially not for clinical proof of principle trials. Clearly, there is a pending need for clinical applicable methodologies that may allow us to quantify, map and monitor hypoxia. Molecular imaging may provide the information required for narrowing the gap between potential and actual patient benefit of hypoxia-targeting strategies. The grand majority of preclinical and clinical work has focused on the usefulness of PET-based assessment of hypoxia-selective tracers. Since hypoxia PET has profound inherent weaknesses, the use of other methodologies, including more indirect methods that quantifies blood flow or oxygenation-dependent flux changes through ATP-generating pathways (eg, anaerobic glycolysis) is being extensively studied. In this review, we briefly discuss established and emerging hypoxia-targeting strategies, followed by a more thorough evaluation of strengths and weaknesses of clinical applicable imaging methodologies that may guide timely treatment intensification to overcome hypoxia-driven resistance. Historically, most evidence for the linkage between hypoxia and poor outcome is based on work in the field of radiotherapy. Therefore, main emphasis in this review is on targeting and imaging of hypoxia for improved radiotherapy.
Clinical imaging of hypoxia: Current status and future directions
2018, Free Radical Biology and Medicine
Tissue hypoxia is a key feature of many important causes of morbidity and mortality. In pathologies such as stroke, peripheral vascular disease and ischaemic heart disease, hypoxia is largely a consequence of low blood flow induced ischaemia, hence perfusion imaging is often used as a surrogate for hypoxia to guide clinical diagnosis and treatment. Importantly, ischaemia and hypoxia are not synonymous conditions as it is not universally true that well perfused tissues are normoxic or that poorly perfused tissues are hypoxic. In pathologies such as cancer, for instance, perfusion imaging and oxygen concentration are less well correlated, and oxygen concentration is independently correlated to radiotherapy response and overall treatment outcomes. In addition, the progression of many diseases is intricately related to maladaptive responses to the hypoxia itself. Thus there is potentially great clinical and scientific utility in direct measurements of tissue oxygenation. Despite this, imaging assessment of hypoxia in patients is rarely performed in clinical settings. This review summarises some of the current methods used to clinically evaluate hypoxia, the barriers to the routine use of these methods and the newer agents and techniques being explored for the assessment of hypoxia in pathological processes.
Biophysical characterization and antineoplastic activity of new bis(thiosemicarbazonato) Cu(II) complexes
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Aiming to explore alternative mechanisms of cellular uptake and cytotoxicity, we have studied a new family of copper(II) complexes (CuL¹-CuL⁴) with bis(thiosemicarbazone) (BTSC) ligands containing pendant protonable cyclic amines (morpholine and piperidine). Herein, we report on the synthesis and characterization of these new complexes, as well as on their biological performance (cytotoxic activity, cellular uptake, protein and DNA binding), in comparison with the parental Cu^IIATSM (ATSM = diacetyl-bis(N4-methylthiosemicarbazonate) complex without pendant cyclic amines. The new compounds have been characterized by a range of analytical techniques including ESI-MS, IR spectroscopy, cyclic voltammetry, reverse-phase HPLC and X-ray spectroscopy. In vitro cytotoxicity studies revealed that the copper complexes are cytotoxic, unlike the corresponding ligands, with a similar potency to that of CuATSM. Unlike CuATSM, the new complexes were able to circumvent cisplatin cross-resistance. The presence of the protonable cyclic amines did not lead to an enhancement of the interaction of the complexes with human serum albumin or calf thymus DNA. However, CuL¹–CuL⁴ showed a remarkably augmented cellular uptake compared with CuATSM, as proved by uptake, internalization and externalization studies that were performed using the radioactive congeners ⁶⁴CuL¹-⁶⁴CuL⁴. The enhanced cellular uptake of CuL¹-CuL⁴ indicates that this new family of Cu^IIBTSC complexes deserves to be further evaluated in the design of metallodrugs for cancer theranostics.
Carbon Nanotubes and Related Nanohybrids Incorporating Inorganic Transition Metal Compounds and Radioactive Species as Synthetic Scaffolds for Nanomedicine Design
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Molecular imaging and applications of nanotechnology in health care are interlinked research areas that are currently of high strategic importance to Europe and worldwide. In this sense, there is great current interest in understanding behavior in cells for target-specific pharmaceuticals assembled for the early detection and therapy of diseases (ranging from cancer to cardiovascular and neurodegenerative diseases). To date, little is understood about the most effective way to assemble and deliver in a targeted manner multimodal contrast agents (here defined as “all in one” imaging probes incorporating metals and metal complexes leading to optical/radiopharmaceuticals and/or paramagnetic nanomaterials) necessary to achieve high-resolution images of processes taking place in cells and tissues, the mechanisms of their uptake in cells and tissues, and the effect that these functional imaging tools have on the targeted cells and tissues. In particular, limited information exists regarding the mechanisms of interactions between functional carbon nanomaterials as new inorganic material-based nanodiagnostics and therapeutic agents for imaging and therapy in diagnostic medicine and cells. A major aspect of this overview that has been virtually unexplored to date in current literature is to rationalize and evaluate the nature and strength of connections among different components of the probe, and to comment on the likely impact on the overall biological functionalities, which is necessary before evaluating their interactions with living systems. In this chapter, different and perhaps unconventional approaches to developing a test-informed protocol for constructing complete bioimaging probes (incorporating inorganic and organometallic transition metal complexes and carbon nanomaterial scaffolds) and testing their functionalities in cells are highlighted. Some approaches to testing and monitoring the delivery, cytotoxicity, and uptake of mechanisms of bioimaging probes assembled on single-walled carbon nanotube scaffolds into a variety of healthy and diseased cells of the complete probes are reviewed. These may be localized on the cells’ surface or inside the cells when derivatized with a targeting group or self-targeted (without a tagged targeting unit).

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: Supported by National Institutes of Health Grants R01 CA84596 and R24 CA83084. ¹⁸F-MISO production supported by Department of Energy Grant DE-FG02-86ER60407. Production and supply of ⁶⁴Cu by Washington University School of Medicine supported by National Cancer Institute Grant R24 CA86307.

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Introduction

Section snippets

Tumor model and experimental design

PET image analysis

Discussion

Int J Radiat Oncol Biol Phys

Radiother Oncol

Int J Radiat Oncol Biol Phys

Appl Radiat Isot

Radiother Oncol

Nucl Med Biol

Int J Radiat Oncol Biol Phys

Nucl Med Biol

The histological structure of some human lung cancers and the possible implications for radiotherapy

Br J Cancer

Oxygenation of human tumorsEvaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements

Cancer Res

Intratumoral pO2 histography as predictive assay in advanced cancer of the uterine cervix

Adv Exp Med Biol

Measurement of human tumour oxygenation status by a polarographic needle electrodeAn analysis of inter- and intratumour heterogeneity

Acta Oncol

Measurement of hypoxia in human tumours by non-invasive SPECT imaging of iodoazomycin arabinoside

Br J Cancer

Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma

Cancer Res

Hypoxia-induced treatment failure in advanced squamous cell carcinoma of the uterine cervix is primarily due to hypoxia induced radiation resistance rather than hypoxia-induced metastasis

Br J Cancer

Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours

Nature

Relationship of hypoxia to metastatic ability in rodent tumours

Br J Cancer

Hypoxia—A key regulatory factor in tumour growth

Nature Rev Cancer

Oxygenation of human tumorsEvaluation of tissue oxygen distribution in breast cancers by computerized O₂ tension measurements

Intratumoral pO₂ histography as predictive assay in advanced cancer of the uterine cervix