We present a non-invasive (albeit contact) method based on Optical Coherence Elastography (OCE) enabling the in vivo segmentation of morphological tissue constituents, in particular, monitoring of ...morphological alterations during both tumor development and its response to therapies. The method uses compressional OCE to reconstruct tissue stiffness map as the first step. Then the OCE-image is divided into regions, for which the Young's modulus (stiffness) falls in specific ranges corresponding to the morphological constituents to be discriminated. These stiffness ranges (characteristic "stiffness spectra") are initially determined by careful comparison of the "gold-standard" histological data and the OCE-based stiffness map for the corresponding tissue regions. After such pre-calibration, the results of morphological segmentation of OCE-images demonstrate a striking similarity with the histological results in terms of percentage of the segmented zones. To validate the sensitivity of the OCE-method and demonstrate its high correlation with conventional histological segmentation we present results obtained in vivo on a murine model of breast cancer in comparative experimental study of the efficacy of two antitumor chemotherapeutic drugs with different mechanisms of action. The new technique allowed in vivo monitoring and quantitative segmentation of (1) viable, (2) dystrophic, (3) necrotic tumor cells and (4) edema zones very similar to morphological segmentation of histological images. Numerous applications in other experimental/clinical areas requiring rapid, nearly real-time, quantitative assessment of tissue structure can be foreseen.
This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of ...photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging. Oxygen content was assessed using phosphorescence lifetime macro-imaging with an oxygen-sensitive probe. For visualization of the perfused microvasculature, an optical coherence tomography-based angiography was used. It was found that PDT induces different alterations in cellular metabolism, depending on the degree of oxygen depletion. Moderate decrease in oxygen in the case of KillerRed was accompanied by an increase in the fraction of free NAD(P)H, an indicator of glycolytic switch, early after the treatment. Severe hypoxia after PDT with Photoditazine resulted from a vascular shutdown yielded in a persistent increase in protein-bound (mitochondrial) fraction of NAD(P)H. These findings improve our understanding of physiological mechanisms of PDT in cellular and vascular modes and can be useful to develop new approaches to monitoring its efficacy.
Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and ...its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response.
Identifying the precise topography of cancer for targeted biopsy in colonoscopic examination is a challenge in current diagnostic practice. For the first time we demonstrate the use of compression ...optical coherence elastography (C-OCE) technology as a new functional OCT modality for differentiating between cancerous and non-cancerous tissues in colon and detecting their morphological features on the basis of measurement of tissue elastic properties. The method uses pre-determined stiffness values (Young's modulus) to distinguish between different morphological structures of normal (mucosa and submucosa), benign tumor (adenoma) and malignant tumor tissue (including cancer cells, gland-like structures, cribriform gland-like structures, stromal fibers, extracellular mucin). After analyzing in excess of fifty tissue samples, a threshold stiffness value of 520 kPa was suggested above which areas of colorectal cancer were detected invariably. A high Pearson correlation (r =0.98; p <0.05), and a negligible bias (0.22) by good agreement of the segmentation results of C-OCE and histological (reference standard) images was demonstrated, indicating the efficiency of C-OCE to identify the precise localization of colorectal cancer and the possibility to perform targeted biopsy. Furthermore, we demonstrated the ability of C-OCE to differentiate morphological subtypes of colorectal cancer - low-grade and high-grade colorectal adenocarcinomas, mucinous adenocarcinoma, and cribriform patterns. The obtained
results highlight prospects of C-OCE for high-level colon malignancy detection. The future endoscopic use of C-OCE will allow targeted biopsy sampling and simultaneous rapid analysis of the heterogeneous morphology of colon tumors.
The oxygen level in a tumor is a crucial factor for its development and response to therapies. Phosphorescence lifetime imaging (PLIM) with the use of phosphorescent oxygen probes is a highly ...sensitive, noninvasive optical technique for the assessment of molecular oxygen in living cells and tissues. Here, we present a protocol for microscopic mapping of oxygen distribution in a mouse tumor model in vivo. We demonstrate that PLIM microscopy, in combination with an Ir(III)-based probe, enables visualization of cellular-level heterogeneity of tumor oxygenation.
Soft biological tissues, breast cancer tissues in particular, often manifest pronounced nonlinear elasticity, i.e., strong dependence of their Young’s modulus on the applied stress. We showed that ...compression optical coherence elastography (C-OCE) is a promising tool enabling the evaluation of nonlinear properties in addition to the conventionally discussed Young’s modulus in order to improve diagnostic accuracy of elastographic examination of tumorous tissues. The aim of this study was to reveal and quantify variations in stiffness for various breast tissue components depending on the applied pressure. We discussed nonlinear elastic properties of different breast cancer samples excised from 50 patients during breast-conserving surgery. Significant differences were found among various subtypes of tumorous and nontumorous breast tissues in terms of the initial Young’s modulus (estimated for stress < 1 kPa) and the nonlinearity parameter determining the rate of stiffness increase with increasing stress. However, Young’s modulus alone or the nonlinearity parameter alone may be insufficient to differentiate some malignant breast tissue subtypes from benign. For instance, benign fibrous stroma and fibrous stroma with isolated individual cancer cells or small agglomerates of cancer cells do not yet exhibit significant difference in the Young’s modulus. Nevertheless, they can be clearly singled out by their nonlinearity parameter, which is the main novelty of the proposed OCE-based discrimination of various breast tissue subtypes. This ability of OCE is very important for finding a clean resection boundary. Overall, morphological segmentation of OCE images accounting for both linear and nonlinear elastic parameters strongly enhances the correspondence with the histological slices and radically improves the diagnostic possibilities of C-OCE for a reliable clinical outcome.
The presence of molecular mutations in colorectal cancer (CRC) is a decisive factor in selecting the most effective first-line therapy. However, molecular analysis is routinely performed only in a ...limited number of patients with remote metastases. We propose to use tissue stiffness as a marker of the presence of molecular mutations in CRC samples. For this purpose, we applied compression optical coherence elastography (C-OCE) to calculate stiffness values in regions corresponding to specific CRC morphological patterns (
= 54). In parallel to estimating stiffness, molecular analysis from the same zones was performed to establish their relationships. As a result, a high correlation between the presence of
/
/
driver mutations and high stiffness values was revealed regardless of CRC morphological pattern type. Further, we proposed threshold stiffness values for label-free targeted detection of molecular alterations in CRC tissues: for
,
, or
driver mutation-above 803 kPa (sensitivity-91%; specificity-80%; diagnostic accuracy-85%), and only for
driver mutation-above 850 kPa (sensitivity-90%; specificity-88%; diagnostic accuracy-89%). To conclude, C-OCE estimation of tissue stiffness can be used as a clinical diagnostic tool for preliminary screening of genetic burden in CRC tissues.
The effects of chemotherapy on the tumor microenvironment remain poorly investigated, although these are crucial for the drug delivery and anti-tumor activity and can modify physiological behavior of ...cancer cells. Optical coherence angiography and phosphorescence lifetime spectroscopy are promising optical methods to characterize both tumor vasculature and its oxygenation, respectively. In this study, we used these methods to explore the influence of irinotecan, a widely used chemotherapeutic agent, on tumor angiogenesis and oxygen content in a mouse model of colorectal cancer in vivo. For the first time, it was shown that irinotecan at conventional dosing reduces tumor oxygenation and blood perfusion via antiangiogenic mechanism of action. Immunohistochemistry for hypoxia and for the endothelial cell marker CD31 verified the in vivo findings. These data show the prospects of the optical techniques to monitor tumor microenvironment in the course of chemotherapy, which can help to optimize and individualize the treatment.
In this paper, we point out some practical obstacles arising in realization of compressional optical coherence elastography (OCE) that have not attracted sufficient attention previously. ...Specifically, we discuss (i) complications in quantification of the Young modulus of tissues related to partial adhesion between the OCE probe and soft intervening reference layer sensor, (ii) distorting influence of tissue surface curvature/corrugation on the subsurface strain distribution mapping, (iii) ways of signal-to-noise ratio (SNR) enhancement in OCE strain mapping when periodic averaging is not realized, and (iv) potentially significant influence of tissue elastic nonlinearity on quantification of its stiffness. Potential practical approaches to mitigate the effects of these complications are also described.
We propose a novel OCT-based method for visualizing microvasculature in three-dimension using reference-free processing of individual complex valued B-scans with highly overlapped A-scans. In the ...lateral direction of such a B-scan, the amplitude and phase of speckles corresponding to vessel regions exhibit faster variability and, thus, can be detected without comparison with other B-scans recorded in the same plane. This method combines elements of several existing OCT angiographic approaches and exhibits: (1) enhanced robustness with respect to bulk tissue motion with frequencies up to tens of Hz, (2) resolution of microcirculation images equal to that of structural images, and (3) possibility of quantifying the vessels in terms of their decorrelation rates.
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