probing-metabolism-viscosity-cancer-cells-using-fluorescence-lifetime

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

Privolzhsky Research Medical University

We studied the effect of laser-induced hydrodynamic on viability of Colo-26 murine colon carcinoma cells in vitro. Laser-induced hydrodynamics was generated by a laser (λ=1.56 μ, power 3 W, 5 min exposure); to this end, the fiber end was submersed into a buffer above the cell monolayer. It was found that laser-induced hydrodynamics destructed the monolayer at standoff distances of between the working end of the laser fiber to cell monolayer of 1 and 5 mm and triggers apoptotic and necrotic death in remaining cells at a distance of 4 mm from the emitter. 

In Vitro Effect of Laser-Induced Hydrodynamics on Cancer Cells.

Ensuring the complete removal of tumor tissue is the main challenge during resection operations. Recently, a technique of "indirect" contact laser surgery has been developed. In this study we assess the possibility of using such surgery for fluorescence image-guided tumor resection. Mouse colon adenocarcinoma CT-26 cells stably expressing the fluorescent protein mKate-2 was used as the tumor model. Resections of the tumor nodes were performed with either a scalpel blade, a laser scalpel with a bare tip, or a laser scalpel with a strongly absorbing coating on the fiber tip. Tumor-positive resection margins were detected using an IVIS Spectrum fluorescence imaging system. After tumor resection with the scalpel blade over half of the animals needed one additional resection to remove residual tumor cells. Animals in this group showed tumor recurrence within 7 days. Fluorescence imaging of the tumor bed, performed after resection to assess the presence of tumor cell clusters, was sufficiently effective only with a bloodless resection. The laser scalpels both with the bare tip and with the strongly absorbing coating on the tip provided such bloodless tumor resection in contact mode. Fewer animals required additional resections when the bare tipped scalpel was used and this also resulted in a reduction in tumor recurrence. After resections were carried out with the laser scalpel with the strongly absorbing coating on the tip, fluorescence was detected in the operative field and this led to undertaking additional resections, although subsequent investigation suggested that this was "false" fluorescence, resulting from the effects of the scalpel rather than the presence of residual tumor cells. The method of laser resection with a strongly absorbing coating on the tip therefore did not appear to demonstrate definite advantages over laser resection with a bare tip when removing tumors.

Can "Indirect" Contact Laser Surgery be Used for Fluorescence-Image Guided Tumor Resections? Preliminary Results.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here  multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

Multiparametric Optical Bioimaging Reveals the Fate of Epoxy Crosslinked Biomeshes in the Mouse Subcutaneous Implantation Model.

Emerging methods of anti-tumor therapies require new approaches to tumor response evaluation, especially enabling label-free diagnostics and  utilization. Here, to assess the tumor early reaction and predict its long-term response, for the first time we apply in combination the recently developed OCT extensions - optical coherence angiography (OCA) and compressional optical coherence elastography (OCE), thus enabling complementary functional/microstructural tumor characterization. We study two vascular-targeted therapies of different types, (1) anti-angiogenic chemotherapy (ChT) and (2) photodynamic therapy (PDT), aimed to indirectly kill tumor cells through blood supply injury. Despite different mechanisms of anti-angiogenic action for ChT and PDT, in both cases OCA demonstrated high sensitivity to blood perfusion cessation. The new method of OCE-based morphological segmentation revealed very similar histological structure alterations. The OCE results showed high correlation with conventional histology in evaluating percentages of necrotic and viable tumor zones. Such possibilities make OCE an attractive tool enabling previously inaccessible  monitoring of individual tumor response to therapies without taking multiple biopsies.

 assessment of functional and morphological alterations in tumors under treatment using OCT-angiography combined with OCT-elastography.

Although fluorescence lifetime imaging microscopy (FLIM) has been extensively applied to study cellular metabolism in the liver, there is neither an established approach to analyze the data, nor have appropriate protocols been developed to maintain the optical metabolic characteristics in the  liver tissue sample. Here, we show that a tri-exponential decay fitting model for the fluorescence signal from nicotinamide adenine dinucleotide (NAD(P)H) and the use of  samples allows the most appropriate processing of the FLIM data. Moreover, we determine the medium that maintains the initial metabolic state of hepatocytes (liver cells), most effectively. Our results should be particularly relevant for the interrogation of liver samples, not only in laboratory research, but also in clinical settings in the future.

Mapping metabolism of liver tissue using two-photon FLIM.

There is a wide range of equivocal melanocytic lesions that can be clinically and dermoscopically indistinguishable from early melanoma. In the present work, we assessed the possibilities of combined using of multiphoton microscopy (MPM) and optical coherence angiography (OCA) for differential diagnosis of the equivocal melanocytic lesions. Clinical and dermoscopic examinations of 60 melanocytic lesions revealed 10 benign lesions and 32 melanomas, while 18 lesions remained difficult to diagnose. Histopathological analysis of these lesions revealed 4 intradermal, 3 compound and 3 junctional nevi in the "benign" group, 7 superficial spreading, 14 lentigo maligna and 11 nodular melanomas in the "melanoma" group and 2 lentigo simplex, 4 dysplastic nevi, 6 melanomas in situ, 4 invasive lentigo melanomas and 2 invasive superficial spreading melanomas in the "equivocal" group. On the basis of MPM, a multiphoton microscopy score (MPMS) has been developed for quantitative assessment of melanoma features at the cellular level, that showed lower score for benign lesions compare with malignant ones. OCA revealed that the invasive melanoma has a higher vessel density and thicker blood vessels than melanoma in situ and benign lesions. Discriminant functions analysis of MPM and OCA data allowed to differentiate correctly between all equivocal melanocytic lesions. Therefore, we demonstrate, for the first time, that a combined use of MPM and OCA has the potential to improve early diagnosis of melanoma.

In vivo multimodal optical imaging of dermoscopic equivocal melanocytic skin lesions.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

Vadim Elagin

Institute of Experimental Oncology and Biomedical Technologies

Privolzhsky Research Medical University

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy