Changes in the tumor oxygenation but not in the tumor volume and tumor vascularization reflect early response of breast cancer to neoadjuvant chemotherapy.

BACKGROUND: Breast cancer neoadjuvant chemotherapy (NACT) allows for assessing tumor sensitivity to systemic treatment, planning adjuvant treatment and follow-up. However, a sufficiently large number of patients fail to achieve the desired level of pathological tumor response while optimal early response assessment methods have not been established now. In our study, we simultaneously assessed the early chemotherapy-induced changes in the tumor volume by ultrasound (US), the tumor oxygenation by diffuse optical spectroscopy imaging (DOSI), and the state of the tumor vascular bed by Doppler US to elaborate the predictive criteria of breast tumor response to treatment. METHODS: A total of 133 patients with a confirmed diagnosis of invasive breast cancer stage II to III admitted to NACT following definitive breast surgery were enrolled, of those 103 were included in the final analysis. Tumor oxygenation by DOSI, tumor volume by US, and tumor vascularization by Doppler US were determined before the first and second cycle of NACT. After NACT completion, patients underwent surgery followed by pathological examination and assessment of the pathological tumor response. On the basis of these, data regression predictive models were created. RESULTS: We observed changes in all three parameters 3 weeks after the start of the treatment. However, a high predictive potential for early assessment of tumor sensitivity to NACT demonstrated only the level of oxygenation, ΔStO2, (ρ = 0.802, p ≤ 0.01). The regression model predicts the tumor response with a high probability of a correct conclusion (89.3%). The "Tumor volume" model and the "Vascularization index" model did not accurately predict the absence of a pathological tumor response to treatment (60.9% and 58.7%, respectively), while predicting a positive response to treatment was relatively better (78.9% and 75.4%, respectively). CONCLUSIONS: Diffuse optical spectroscopy imaging appeared to be a robust tool for early predicting breast cancer response to chemotherapy. It may help identify patients who need additional molecular genetic study of the tumor in order to find the source of resistance to treatment, as well as to correct the treatment regimen.

Special Section Guest Editorial: Topical Problems of Biophotonics: from Optical Bioimaging to Clinical Biophotonics.

This editorial provides an introductory overview for the Special Section on Topical Problems of Biophotonics.

Combination of virtual point detector concept and fluence compensation in acoustic resolution photoacoustic microscopy.

We propose a hybrid approach to image enhancement in acoustic resolution photoacoustic microscopy. The developed technique is based on compensation for nonuniform spatial sensitivity of the optoacoustic (OA) system in both optical and acoustic domains. Spatial distribution of optical fluence is derived from full three-dimensional Monte Carlo simulations accounting for conical geometry of tissue laser illumination at the wavelength of 532 nm. Approximate nonuniform spatial response of acoustic detector with numerical aperture of 0.6 is derived from the two-dimensional k-Wave modeling. Application of the developed technique allows to improve the spatial resolution and to balance in-depth signal-level distribution in OA images of phantom and in-vivo objects.

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination.

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells.

BACKGROUND: Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS: HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS: Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE: This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice.

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Lifetime imaging of FRET between red fluorescent proteins.

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

Comparative study of tumor hypoxia by diffuse optical spectroscopy and immunohistochemistry in two tumor models.

The capabilities of diffuse optical spectroscopy for noninvasive assessing of oxygen status in experimental tumors have been demonstrated. Specific features of the distribution of total hemoglobin, oxygenated hemoglobin, deoxygenated hemoglobin, and blood-oxygen saturation were shown on two tumor models having different histological structure and functional characteristics. The results obtained by the optical technique were verified by immunohistochemical study of tissue samples marked with exogenous marker of hypoxia--pimonidazole.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment.

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Fluorescence diffuse tomography for detection of red fluorescent protein expressed tumors in small animals.

A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.

Novel algorithm of processing optical coherence tomography images for differentiation of biological tissue pathologies.

A numerical algorithm based on a small-angle approximation of the radiative transfer equation (RTE) is developed to reconstruct scattering characteristics of biological tissues from optical coherence tomography (OCT) images. According to the algorithm, biological tissue is considered to be a layered random medium with a set of scattering parameters in each layer: total scattering coefficient, variance of a small-angle scattering phase function, and probability of backscattering, which fully describe the OCT signal behavior versus probing depth. The reconstruction of the scattering parameters is performed by their variation to fit the experimental OCT signal by the theoretical one using a time-saving genetic algorithm. The proposed reconstruction procedure is tested on model media with known scattering parameters. The possibility to estimate scattering parameters from OCT images is studied for various regimes of OCT signal decay. The developed algorithm is applied to reconstruct optical characteristics of epithelium and stroma for normal cervical tissue and its pathologies, and the potential to distinguish between the types of pathological changes in epithelial tissue by its OCT images is demonstrated.

Optical coherence tomography for in situ monitoring of laser corneal ablation.

OBJECTIVE: To improve the precision of refractive surgery, a new approach for determination of the removed corneal thickness profile in situ with laser ablation by optical coherence tomography (OCT) is developed. STUDY DESIGN/MATERIALS AND METHODS: The traditional method for precision (less than 10 microm) measurements of intraocular distances is based on the use of the reflected component of probing radiation. This component is characterized by a small range of operating angles between a probing beam and a normal to the surface under study. To enhance this range of operating angles we suggest using a light component backscattered from a biological object. This will enable precision measurements over the entire surface of the cornea without any changes in the orientation between a probing beam and the eye, a necessary condition for in situ monitoring of laser refraction correction in the eye. We suggest a specially developed algorithm of OCT signal processing to measure the corneal thickness by the backscattered light component for a single longitudinal scan (A scan). The corneal thickness profile is obtained by a series of such A scans acquired by successively scanning a probing beam along the corneal surface. The thickness profile of removed layer is determined by changes in the corneal thickness profile in the process of ablation. When the cornea is ablated by a beam with a fixed transverse profile, we propose using integral characteristics of the ablated layer profile, for example, the maximum ablation depth, as criteria of changes in refractive power of the eye. The measurement precision by these characteristics is considerably higher than by a single A scan. Since the cornea is a poorly scattering medium, the Fourier filtering is employed to increase reliability and precision of the method. Model experiments on monitoring the ablation process in a lavsan film and ex vivo human cornea are described. Preliminary experiments on in vivo measurements of human corneal thickness are performed. RESULTS: In model experiments the precision of measurement of laser ablation depth by one A scan was 5-20 microm, depending on the signal-to-noise ratio (SNR), whereas the precision of measurement of laser ablation depth as the integral characteristic of the ablated layer profile was 0.3-5 microm. The experimental results showed that at small SNR Fourier filtering might considerably increase reliability and precision of measurements. When SNR is high, the measurement precision does not change. The precision of measurements of the corneal thickness in preliminary in vivo experiments was higher than in ex vivo experiments. This factor is very promising for application of the method suggested herein in refractive surgery.