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The major goal in developing drugs targeting specific tumor receptors, such as Monoclonal AntiBodies (MAB), is to make a drug compound that targets selectively the cancer-causing biomarkers, inhibits their functionality, and/or delivers the toxin specifically to the malignant cells. Recent advances in MABs show that their efficacy depends strongly on characterization of tumor biomarkers. Therefore, one of the main tasks in cancer diagnostics and treatment is to develop non-invasive in-vivo imaging techniques for detection of cancer biomarkers and monitoring their down regulation during the treatment. Such methods can potentially result in a new imaging and treatment paradigm for cancer therapy. In this article we have reviewed fluorescence imaging approaches, including those developed in our group, to detect and monitor Human Epidermal Growth Factor 2 (HER2) receptors before and during therapy. Transition of these techniques from the bench to bedside is the ultimate goal of our project. Similar approaches can be used potentially for characterization of other cancer related cell biomarkers.

Two noninvasive methods, thermography and laser Doppler imaging (LDI), were assessed for their ability to quantitatively assess parameters of vascularity in lesions of HIV-associated Kaposi's sarcoma (KS). Thermography and LDI images of a representative KS lesion were recorded in 16 patients and compared to normal skin either adjacent to the lesion or on the contralateral side. Eleven of the 16 patients had greater than 0.5 °C increased temperature and 12 of the 16 patients had increased flux (measured by LDI) as compared to normal skin. There was a strong correlation between these two parameters (R = 0.81, p < 0.001). In ten patients, measurements were obtained prior to therapy and after receiving a regimen of liposomal doxorubicin and interleukin-12. After 18 weeks of therapy, temperature and blood flow of the lesions were significantly reduced from the baseline (p = 0.004 and 0.002 respectively). These techniques hold promise to assess physiologic parameters in KS lesions and their changes with therapy.

Optical methods have a long history in the field of medical diagnosis. The biomolecular specificity possible with optical methods has been particularly valuable in microscopy and histopathology while in vivo imaging of deep structures has traditionally been the domain of X-ray and MRI. The use of optical methods in deep tissue has been limited by multiple-scattering which blurs or distorts the optical signal. New stochastic methods which account for multiple scattering have been developed that are extending the usefulness of optical methods deep into tissue. In optical mammography, photons may travel through 10cm of tissue before arriving at the detector. We have developed a method for quantifying parameters of anomalous sites in breast tissue that may be used for functional characterization of tumors. In other work presented here, we are developing fluorescence based methods to detect and monitor tumor status. The immune response to a tumor is a target for fluorescently labeled specific antibodies. We have developed a method to localize the tumor site using CW fluorescence. Additionally, we have developed a method which uses time-resolved data and capitalizes on probe lifetime sensitivity to metabolic parameters such as pH and temperature to obtain functional information from the tumor site.

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain–fused-(Z HER2:342)2 -Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA]) that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor). The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.