Imaging is ideally suited to measuring in vivo tumor biology related to basic physiological properties such as perfusion, metabolism, biosynthesis, cell proliferation, and cell death. In fact, many of these processes are difficult to assay by tissue sampling; therefore, imaging provides a unique and quantitative measure of these properties that can only be measured in vivo.

Tumor perfusion is one of the earliest physiological properties to be measured, and advances in methodology have led to increasingly quantitative approaches. The most physiologically robust and quantitative measures of tumor blood flow use freely diffusible imaging probes, where blood flow can be inferred from the time course of probe uptake and washout, adapting methods developed for measuring cerebral blood flow 37. One example is the use of ¹⁵O-water to measure tumor blood flow by PET, which yields measures of tumor blood flow in ml/minute/g that have been validated against microsphere methods 38. Water PET imaging has been shown to be effective in monitoring breast cancer response, and changes in tumor perfusion with treatment measured by water PET are predictive of survival 39. Diffusable optical probes may also be used for this purpose 29. Considerable effort has been devoted to the quantification of tumor perfusion and tumor capillary permeability using radiographic and MRI contrast agents 4041424344. These agents have somewhat limited permeability across capillaries; therefore, their in vivo kinetics are dependent upon both blood flow and capillary permeability. Increasingly sophisticated image acquisition and analysis methods, for DCE-MRI in particular, have led to the ability to measure regional breast cancer perfusion and capillary permeability in both animal models and patients 17. Novel MRI contrast agents, including macromolecular agents, may offer additional capabilities 45. These methods have been applied in early trials of anti-angiogenic therapy breast cancer and have yielded insights into the nature of response 16. Standardized methods for DCE-MRI for clinical trials have been proposed 46.

Tumor perfusion imaging requires good vascular access for prompt and reliable delivery of the imaging probe, and has therefore been more challenging in animal models; however, recent work using inhaled radionuclide agents hold promise for small animal research applications 47.

Tumor metabolism has also been widely studied by imaging. Perhaps the best known example is the measurement of regional glucose metabolism using ¹⁸F-fluorodeoxyglucose (FDG) PET, which is now routinely used in breast cancer clinical practice to determine the extent of tumor spread and assess response to treatment 525. This method can also be readily applied to animal models 48, and can yield detailed physiological information through kinetic analysis of dynamic images 4950. Other PET radiopharmaceuticals can be used to measure other aspects of metabolism, including regional oxygen consumption and fatty acid metabolism 25. MRS has also been quite useful in measuring metabolism through ¹H spectroscopy to measure levels of lactate and other biochemical species key in metabolism 51 and also through phosphorous spectroscopy to measure concentrations of adenine nucleotides as an indicator of energy metabolism 52. Other studies in a variety of tumors, especially in brain tumors and prostate cancer, have shown the ability to measure biochemical species in a variety of metabolic pathways – for example, membrane lipids (choline) and metabolic intermediates (citrate) – and have provided improved diagnosis, early measures of response, and insights into pathophysiology 535455565758. The chemical composition of the breast, particularly its high lipid content, provides some challenges for MRS, but recent progress in magnet field strength, acquisition, and processing methods may offer improved capabilities in breast cancer 59.

Aberrant cellular proliferation is a fundamental property of cancer, including breast cancer 60. Labeled compounds such as ¹⁴C- or ³H-thmyidine have been an important method for measuring cellular proliferation through sampling, dating back over 40 years 61. Early work used PET and ¹¹C-thymidine to measure tumor proliferation by imaging, and quantitative imaging approaches were validated against in vitro assay gold standards 62. However, the short half-life of ¹¹C (20 minutes) and the extensive in vivo metabolism of thymidine limit the feasibility of this approach for both animal and patient imaging. More recent work using thymidine analogs labeled with ¹⁸F (half-life 109 minutes) have been developed and undergone considerable advances in recent years 6364. The most promising of these had been ¹⁸F-fluorothymidine (FLT), with notable recent results in both animal and patient breast cancer imaging 65666768. FLT PET appears especially promising for measuring the early effects of therapy on breast cancer growth, as suggested by recent studies by Kenny and colleagues 66. This imaging approach had also been validated against in vitro assay of proliferation (reviewed in 63) and is poised for greater use in both animal research and human clinical trials.

Methods for imaging cell death have also been investigated, but are at an earlier stage of development. Many of these have been based upon an extension of Annexin V staining in vitro, which indicates apoptotic cells through binding to phosphotidyl serines 69. The molecules are found only on the inner surface of plasma membranes and, therefore, normally not accessible to Annexin V, a peptide, for binding. However, during apoptosis, these molecules are transiently exposed to the extracellular space, allowing binding of Annexin 69. The earliest studies used ^99mTc-annexin and SPECT imaging to measure apoptosis in animal models and patients 6970. More recently, methods for annexin-based apoptosis imaging have been developed for PET, MRI, optical imaging, and ultrasound 71. One limitation of this approach has been the transient nature of phosphatidyl serine exposure during cell death, resulting in fairly limited signal for imaging 72. Other approaches targeted to other aspects of the apoptotic cascade are being investigated 71. An alternative, but less specific approach has been to use MRI measures of water diffusion through the extracellular space as an indirect measure of tumor cellularity 1973. Increases in the diffusion coefficient, as an indicator of a decrease in tumor cellularity, have correlated with measures of apoptosis in animal models 19 and response to therapy in early patient studies 2073. This method has the advantage of being available using existing MRI instrumentation for both animal and patient imaging without the need for imaging probes, but provides a relatively indirect measure of cell death.

Several methods can also measure biosynthesis as an indicator of tumor growth, with approaches targeted to protein synthesis and membrane synthesis. The uptake of labeled amino acids, such as ¹¹C-methionine, has been shown to correlate with tumor growth, and changes in uptake provide an early indication of breast cancer response to therapy 74. This approach, however, is limited by the complex nature of amino acid metabolism pathways, making it difficult to measure protein synthetic rate versus amino acid transport and metabolism 75. Artificial amino acids have also been tested as indicators of amino acid transport 76.

Proliferating tumor cells also engage in enhanced lipid biosynthesis to provide material needed for cellular membranes 77. This process can also be assayed through molecular imaging using several different methods. Spurred by results in brain tumor imaging 57, MRS studies of breast cancer have shown increased choline pool sizes to be a feature of breast malignancy 978. Interestingly, changes in the choline concentration measured by MRS early in treatment appear to be a marker for early response to therapy, as early as 24 hours after treatment with chemotherapy 22. These exciting early findings are now being tested in a large prospective cooperative group trial. Lipid metabolism can also be studied by PET using either ¹¹C or ¹⁸F labeled choline, or ¹¹C-acetate 79, which enters lipid synthesis from the tricarboxylic acid (TCA) cycle via fatty acid synthetase. Fatty acid synthetase has been show to have increased activity and expression in cancer and may be a target for therapy 80. This approach has shown considerable promise in other tumors such as prostate cancer 81, including therapeutic response 82, but has not been applied to the same extent in breast cancer 83.

The ability to measure the expression of specific proteins that are gene products associated with breast cancer has led to important advances in breast cancer treatment. Examples include the expression of estrogen receptors (ERs), a target for endocrine therapy 84, and HER2, also increasingly a target of tumor-specific treatment 85. Molecular imaging has also been applied to measuring specific protein expression 624. Advantages of imaging include its non-invasiveness, the ability to measure receptor expression in the entire disease burden and thus the ability to avoid sampling error that can occur with heterogeneous receptor expression, and the potential for serial studies of in vivo drug effects on the target. A very practical consideration is that imaging can assess receptor expression at sites that are challenging to sample and assay, for example, bone metastases, where de-calcification can make assay of tumor gene products challenging.

Imaging protein expression, particularly tumor receptors, poses some unique challenges. For receptors, imaging results can be quite sensitive to the molecular quantity of the imaging probe needed to generate the image. Most receptors have high affinity for their ligands and are active at micromolar or nanomolar concentrations of the ligand. Even small molar quantities of the imaging agent may saturate the receptor and limit the ability to visualize receptor expression 8687. For this reason, molecular imaging of tumor receptors has been most successful to date with radionuclide imaging, PET and SPECT, where it is possible to generate images with nanomolar or picomolar amounts of the imaging probe. For larger molecules, like peptides and monoclonal antibodies, other labels suitable for optical, MR, and ultrasound imaging are possible 6; however, for small-molecule receptor imaging agents, such as labeled steroids for steroid receptors, radionuclide imaging appears to be the only feasible approach.

The most work to date in this area of breast cancer research has been done for steroid receptors 688. Considerable efforts have gone into the development of radiopharmaceuticals for ER imaging, as reviewed in 888990. Although a variety of ER imaging agents have been tested, and continue to be developed and tested, the most successful ER imaging radiopharmaceutical to date is 16 alpha-[¹⁸F]-fluoro-17 beta-estradiol (FES) 8791. FES has binding characteristics similar to estradiol for both the ER and the transport protein SHBG 9293. It can be synthesized with sufficient specific activity that high-quality patient images can be made with injections of less than 5 μg of FES 94. Regional estrogen binding is readily quantified by FES PET, and FES uptake has been validated as a measure of ER expression in breast tumors against ER expression assay of tissue samples by radioligand binding 95 and immunohistochemistry 96. FES uptake is readily visualized and quantified in primary and metastatic breast cancer 97. It can identify heterogeneous ER expression, for example, loss of ER expression in metastases arising from ER-expressing primary tumors 9497. The level of FES uptake has been shown to be predictive of response to endocrine therapy 9498, including heavily pre-treated patients (Figure 1). Serial FES PET can also measure the pharmacodynamic effect of drugs on estradiol binding to the ER 9899, yielding insights into determinants of drug efficacy.

While PET ER imaging had been successful, efforts to image progesterone receptor (PR) have been less successful 100101. In a study of the PR imaging agent 21-[¹⁸F]fluoro-16 alpha-ethyl-19-norprogesterone, uptake was seen in some tumors, but the level of uptake did not correlate with the level of PR expression. This may be in large part due to the relatively low affinity of progestins for the PR, with binding affinities that are orders of magnitude lower than those of androgens and androgen receptors and estrogens and ERs 101. As such, relatively high non-specific binding compared to specific binding of imaging probes may limit their utility for PR imaging. Later studies also showed that the radiopharmaceutical tested was rapidly metabolized in human to a metabolite with poor receptor binding 102, a finding that was not predicted by pre-clinical models. Efforts to develop effective PR imaging agents continue 103.

HER2 (ErbB2) expression in breast cancer has become an important indicator of prognosis and an increasingly important target for therapy 104. Recent efforts have focused on imaging HER2 expression in breast cancer. The most success and largest number of studies to date used imaging probes based upon immune recognition to image HER2 expression. Specific imaging probes based on radiolabeled antibodies or fragments 105106107108109, or novel constructs such as affibodies 110111, have shown success in early studies. Studies using a ⁶⁸Ga-labeled F(ab')2 fragment of trastuzumab by Smith-Jones and colleagues 112113 demonstrated the feasibility of measuring regional HER2 expression in murine animal models. The imaging results nicely demonstrated alterations in HER2 expression accompanying experimental therapy using HSP90-directed agents (geldamycin analogs) to disrupt protein chaperoning and reduce HER2 expression 112113. Studies using ¹³¹I- or ¹¹¹In-labeled trastuzumab have demonstrated the ability to image tumor expression of HER2 and tumor and normal tissue accumulation of trastuzumab 108114115, although there has been some controversy about the significance of uptake in normal tissues prone to trastuzumab toxicity, such as the heart 108115. Promising early patient studies have also been presented for ⁸⁹Zr-labeled trastuzumab 116.

For imaging using larger molecules like monoclonal antibodies and fragments, as in the case of HER2 imaging, an even wider range of probes (and therefore imaging modalities) may be possible, including optical, ultrasound, and MR-based probes 117118119120121122. Koyama 123 demonstrated specific fluorescence imaging of HER2-expressing lung metastasis in an animal model. Others have used near-infrared imaging of HER2 expression in vitro using gold nanoshell bioconjugates 124 and fluorochrome labeling 125. Pre-clinical studies using specific antibodies conjugated to gadolinium or magnetic nanoparticles demonstrated the feasibility of MRI antibody imaging in cells 126 and animal models 118. Early studies of nanoparticle-based ultrasound probes conjugated to HER2-specific antibodies have demonstrated the feasibility of this approach in early in vitro studies and simulated in vivo studies 119127. The ability to image with multiple modalities is particularly helpful for translating research from the pre-clinical to clinical setting. For example, optical imaging is extremely valuable for small-animal studies, but has limited utility in patients due to poor tissue penetration for imaging deeper structures. The ability to translate optical imaging findings into PET or MRI imaging in patients, seen in early work for HER2 imaging, holds great promise for facilitating translational research.

Molecular imaging provides a unique opportunity to image the tumor microenvironment, which is challenging by more invasive means. Tumor hypoxia, an important factor mediating cancer aggressiveness and therapeutic resistance 128129, has been widely studied by imaging, with some recent studies in breast cancer 130. Most work has been done using PET imaging and the agent ¹⁸F-fluoromisonidazole (FMISO) (Figure 2); however, other PET hypoxia probes have been developed and tested 38. Other hypoxia imaging methods based upon MRI and optical approaches are at an earlier stage of development, but appear promising 131132.

Tumor vasculature plays a key role in tumor growth and metastasis, and is also important in the delivery of systemic therapeutic agents. Imaging of tumor vasculature and the delivery of nutrients and drugs has also been an area of interest. Tumor perfusion and capillary transport provide an indirect measure of tumor vasculature and can be imaged by DCE-MRI and PET, which have been effective in measuring tumor response in early studies 163839. In addition, targeted imaging probes can non-invasively and specifically assess tumor neo-vasculature. PET probes based upon specific labeled peptides that bind to integrins expressed in neovessels have been studied in animals and tested in humans 133134. MRI probes have been developed and are at an earlier stage of testing 40. Such agents may be especially helpful for therapies directed at tumor neo-vasculature, such as bevacizumab.

Imaging methods to measure nutrient and drug transport have also been developed and tested. For example, the SPECT agent ^99mTc-sestamibi, developed for myocardial perfusion imaging, has been shown to be a substrate for the drug efflux transporter P-glycoprotein 135. Studies in the setting of pre-surgical chemotherapy showed that the uptake and retention of ^99mTc-sestamibi was predictive of response to chemotherapeutic agents that are substrates for P-glycoprotein, such as epirubicin 136. Somewhat more specific and quantitative probes of P-glycoprotein transport have been developed for PET imaging 137138139. Macromolecular MRI contrast agents are undergoing testing in patients 140 and may be useful for measuring the delivery of therapeutic agents, such as trastuzumab, that are large molecules where capillary and interstitial transport may pose significant barriers.

Advances in cancer biology have led to an increasing array of approaches to breast cancer therapy, including immunotherapy and gene therapy. These advances in turn lead to the need for increasingly sophisticated approaches for imaging to monitor these treatments. An increasing body of work in molecular imaging has therefore been devoted to imaging approaches capable of imaging gene expression and cellular trafficking and survival in vivo 1282.

Paralleling work done in reporter systems for cell culture, imaging reporter systems have been developed and are increasingly used in animal model research 141142143.

Reporter imaging can use optical approaches, with reporter genes expressing optically detectable molecules such as green fluorescent protein and firefly luciferase. Optical approaches provide inexpensive, readily available methods for imaging reporter gene expression, and are widely used in current research 144. Radionuclide methods have also been developed and widely tested in animal models 143. The most popular approach has been based upon the use of viral thymidine kinase as a reporter gene, combined with labeled probes that are specific substrates for phosphorylation by viral thymidine kinase 145146. This results in probe trapping and high-contrast, readily quantifiable images 147. A variety of other radionuclide reporter systems have been used, including the NaI symporter and radioiodine, receptor expression (for example, dopamine) and receptor binding ligands, and alternative metabolic substrates 148149. Some progress has also been made in MRI reporter systems 150151; however, this work is at an earlier stage than optical and PET approaches.

The development of therapeutic approaches mediated by specific cells – for example, immunotherapy – has generated increasing interest in cell labeling and imaging. Cell labeling can be done using non-specific probes, such as ¹¹¹In-oxine and non-targeted iron oxide particles for use in SPECT and MRI, respectively 152; however, this approach often requires isolation of the cells to assure that only the desired cells are imaged. In cell therapy where molecular manipulation is possible, cells engineered with reporter systems can also be used to provide specific cell labeling that does not require isolation of cells and that remains with the cells through subsequent generations, unlike most non-specific methods 153. For immunotherapy, the generation of reporter systems that do not themselves engender an immune response is a challenge and represents an active area of research 154.