Tissue Elasticity as a Diagnostic Marker of Molecular Mutations in Morphologically Heterogeneous Colorectal Cancer.

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 (n = 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 KRAS/NRAS/BRAF 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 KRAS, NRAS, or BRAF driver mutation-above 803 kPa (sensitivity-91%; specificity-80%; diagnostic accuracy-85%), and only for KRAS 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.

Oxygen Assessment in Tumors In Vivo Using Phosphorescence Lifetime Imaging Microscopy.

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.

Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

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.

Depth-Resolved Attenuation Mapping of the Vaginal Wall under Prolapse and after Laser Treatment Using Cross-Polarization Optical Coherence Tomography: A Pilot Study.

Vaginal wall prolapse is the most common type of pelvic organ prolapse and is mainly associated with collagen bundle changes in the lamina propria. Neodymium (Nd:YAG) laser treatment was used as an innovative, minimally invasive and non-ablative procedure for the treatment of early-stage vaginal wall prolapse. The purpose of this pilot study was to assess connective tissue changes in the vaginal wall under prolapse without treatment and after Nd:YAG laser treatment using cross-polarization optical coherence tomography (CP OCT) with depth-resolved attenuation mapping. A total of 26 freshly excised samples of vaginal wall from 26 patients with age norm (n = 8), stage I-II prolapses without treatment (n = 8) and stage I-II prolapse 1-2 months after Nd:YAG laser treatment (n = 10) were assessed. As a result, for the first time, depth-resolved attenuation maps of the vaginal wall in the B-scan projection in the co- and cross-polarization channels were constructed. Two parameters within the lamina propria were target calculated: the median value and the percentages of high (≥4 mm-1) and low (<4 mm-1) attenuation coefficient values. A significant (p < 0.0001) decrease in the parameters in the case of vaginal wall prolapse compared to the age norm was identified. After laser treatment, a significant (p < 0.0001) increase in the parameters compared to the normal level was also observed. Notably, in the cross-channel, both parameters showed a greater difference between the groups than in the co-channel. Therefore, using the cross-channel achieved more reliable differentiation between the groups. To conclude, attenuation coefficient maps allow visualization and quantification of changes in the condition of the connective tissue of the vaginal wall. In the future, CP OCT could be used for in vivo detection of early-stage vaginal wall prolapse and for monitoring the effectiveness of treatment.

Multimodal OCT Control for Early Histological Signs of Vulvar Lichen Sclerosus Recurrence after Systemic PDT: Pilot Study.

Photodynamic therapy (PDT) is a modern treatment for severe or treatment-resistant vulvar lichen sclerosus (VLS). The chronic and recurrent nature of VLS requires control of recurrences at an early stage. In this paper, a non-invasive multimodal optical coherence tomography (OCT) method was used to control for early histological signs of VLS recurrence after systemic PDT using Photodithazine®. To interpret the OCT data, a histological examination was performed before PDT and 3 months after PDT. Two groups of patients were identified: with early histological signs of VLS recurrence (Group I, n = 5) and without histological signs of VLS recurrence (Group II, n = 6). We use structural OCT, OCT angiography, and OCT lymphangiography throughout 6 months after PDT to visually assess the skin components and to quantitatively assess the dermis by calculating the depth-resolved attenuation coefficient and the density of blood and lymphatic vessels. The OCT data assessment showed a statistically significant difference between the patient groups 3 months after PDT. In Group II, all the studied OCT parameters reached maximum values by the 3rd month after PDT, which indicated recovery of the skin structure. At the same time, in Group I, the values of OCT parameters did not approach the values those in Group II even after 6 months. The obtained results of multimodal OCT can be used for non-invasive control of early histological recurrence of VLS after systemic PDT and for adjusting treatment tactics in advance, without waiting for new clinical manifestations of the disease.

Depth-resolved method for attenuation coefficient calculation from optical coherence tomography data for improved biological structure visualization.

Optical coherence tomography (OCT) is a promising tool for intraoperative tissue morphology determination. Several studies suggest that attenuation coefficient derived from the OCT images, can differentiate between tissues of different morphology, such as normal and pathological structures of the brain, skin, and other tissues. In the present study, the depth-resolved method for attenuation coefficient calculation was adopted for the real-world situation of the depth-dependent OCT sensitivity and additive imaging noise with nonzero mean. It was shown that in the case of sharp focusing (~10 µm spot full width at half maximum [FWHM] or smaller at 1.3 µm central wavelength) only the proposed method for depth-dependent sensitivity compensation does not introduce misleading artifacts into the calculated attenuation coefficient distribution. At the same time, the scanning beam focus spot with FWHM greater than 10 µm at 1.3 µm central wavelength allows one to use multiple approaches to the attenuation coefficient calculation without introducing noticeable bias. This feature may hinder the need for robust corrections for the depth-resolved attenuation coefficient estimations from the community.

Compression OCT-elastography combined with speckle-contrast analysis as an approach to the morphological assessment of breast cancer tissue.

Currently, optical biopsy technologies are being developed for rapid and label-free visualization of biological tissue with micrometer-level resolution. They can play an important role in breast-conserving surgery guidance, detection of residual cancer cells, and targeted histological analysis. For solving these problems, compression optical coherence elastography (C-OCE) demonstrated impressive results based on differences in the elasticity of different tissue constituents. However, sometimes straightforward C-OCE-based differentiation is insufficient because of the similar stiffness of certain tissue components. We present a new automated approach to the rapid morphological assessment of human breast cancer based on the combined usage of C-OCE and speckle-contrast (SC) analysis. Using the SC analysis of structural OCT images, the threshold value of the SC coefficient was established to enable the separation of areas of adipose cells from necrotic cancer cells, even if they are highly similar in elastic properties. Consequently, the boundaries of the tumor bed can be reliably identified. The joint analysis of structural and elastographic images enables automated morphological segmentation based on the characteristic ranges of stiffness (Young's modulus) and SC coefficient established for four morphological structures of breast-cancer samples from patients post neoadjuvant chemotherapy (residual cancer cells, cancer stroma, necrotic cancer cells, and mammary adipose cells). This enabled precise automated detection of residual cancer-cell zones within the tumor bed for grading cancer response to chemotherapy. The results of C-OCE/SC morphometry highly correlated with the histology-based results (r =0.96-0.98). The combined C-OCE/SC approach has the potential to be used intraoperatively for achieving clean resection margins in breast cancer surgery and for performing targeted histological analysis of samples, including the evaluation of the efficacy of cancer chemotherapy.

Intraoperative Assessment of Breast Cancer Tissues after Breast-Conserving Surgery Based on Mapping the Attenuation Coefficients in 3D Cross-Polarization Optical Coherence Tomography.

Intraoperative differentiation of tumorous from non-tumorous tissue can help in the assessment of resection margins in breast cancer and its response to therapy and, potentially, reduce the incidence of tumor recurrence. In this study, the calculation of the attenuation coefficient and its color-coded 2D distribution was performed for different breast cancer subtypes using spectral-domain CP OCT. A total of 68 freshly excised human breast specimens containing tumorous and surrounding non-tumorous tissues after BCS was studied. Immediately after obtaining structural 3D CP OCT images, en face color-coded attenuation coefficient maps were built in co-(Att(co)) and cross-(Att(cross)) polarization channels using a depth-resolved approach to calculating the values in each A-scan. We determined spatially localized signal attenuation in both channels and reported ranges of attenuation coefficients to five selected breast tissue regions (adipose tissue, non-tumorous fibrous connective tissue, hyalinized tumor stroma, low-density tumor cells in the fibrotic tumor stroma and high-density clusters of tumor cells). The Att(cross) coefficient exhibited a stronger gain contrast of studied tissues compared to the Att(co) coefficient (i.e., conventional attenuation coefficient) and, therefore, allowed improved differentiation of all breast tissue types. It has been shown that color-coded attenuation coefficient maps may be used to detect inter- and intra-tumor heterogeneity of various breast cancer subtypes as well as to assess the effectiveness of therapy. For the first time, the optimal threshold values of the attenuation coefficients to differentiate tumorous from non-tumorous breast tissues were determined. Diagnostic testing values for Att(cross) coefficient were higher for differentiation of tumor cell areas and tumor stroma from non-tumorous fibrous connective tissue: diagnostic accuracy was 91-99%, sensitivity-96-98%, and specificity-87-99%. Att(co) coefficient is more suitable for the differentiation of tumor cell areas from adipose tissue: diagnostic accuracy was 83%, sensitivity-84%, and specificity-84%. Therefore, the present study provides a new diagnostic approach to the differentiation of breast cancer tissue types based on the assessment of the attenuation coefficient from real-time CP OCT data and has the potential to be used for further rapid and accurate intraoperative assessment of the resection margins during BCS.

Towards targeted colorectal cancer biopsy based on tissue morphology assessment by compression optical coherence elastography.

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 ex vivo 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.

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor.

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.

Compression optical coherence elastography versus strain ultrasound elastography for breast cancer detection and differentiation: pilot study.

The aims of this study are (i) to compare ultrasound strain elastography (US-SE) and compression optical coherence elastography (C-OCE) in characterization of elastically linear phantoms, (ii) to evaluate factors that can cause discrepancy between the results of the two elastographic techniques in application to real tissues, and (iii) to compare the results of US-SE and C-OCE in the differentiation of benign and malignant breast lesions. On 22 patients, we first used standard US-SE for in vivo assessment of breast cancer before and then after the lesion excision C-OCE was applied for intraoperative visualization of margins of the tumors and assessment of their type/grade using fresh lumpectomy specimens. For verification, the tumor grades and subtypes were determined histologically. We show that in comparison to US-SE, quantitative C-OCE has novel capabilities due to its ability to locally control stress applied to the tissue and obtain local stress-strain curves. For US-SE, we demonstrate examples of malignant tumors that were erroneously classified as benign and vice versa. For C-OCE, all lesions are correctly classified in agreement with the histology. The revealed discrepancies between the strain ratio given by US-SE and ratio of tangent Young's moduli obtained for the same samples by C-OCE are explained. Overall, C-OCE enables significantly improved specificity in breast lesion differentiation and ability to precisely visualize margins of malignant tumors compared. Such results confirm high potential of C-OCE as a high-speed and accurate method for intraoperative assessment of breast tumors and detection of their margins.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

Nonlinear Elasticity Assessment with Optical Coherence Elastography for High-Selectivity Differentiation of Breast Cancer Tissues.

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.

Lymph vessels visualization from optical coherence tomography data using depth-resolved attenuation coefficient calculation.

Multimodal optical coherent tomography grows popularity with researchers and clinicians over the past decade. One of the modalities is lymphangiography, which allows visualization of the lymphatic vessel networks within optical coherence tomography (OCT) imaging volume. In the present study, it is shown that lymphatic vessel visualization obtained from the depth-resolved attenuation coefficient distributions, corrected for the noise, shows improved contrast and detail in comparison with previously proposed approaches. We also argue that the two most popular approaches for lymphatic vessel visualization, namely simple intensity thresholding and vesselness calculation based on local Hessian matrix eigenvalues, imply different definitions of the lymphatic vessel's appearance in the OCT volume and lead to the different networks.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

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.

Diagnostic Accuracy of Cross-Polarization OCT and OCT-Elastography for Differentiation of Breast Cancer Subtypes: Comparative Study.

The possibility to assess molecular-biological and morphological features of particular breast cancer types can improve the precision of resection margin detection and enable accurate determining of the tumor aggressiveness, which is important for treatment selection. To enable reliable differentiation of breast-cancer subtypes and evaluation of resection margin, without performing conventional histological procedures, here we apply cross-polarization optical coherence tomography (CP-OCT) and compare it with a novel variant of compressional optical coherence elastography (C-OCE) in terms of the diagnostic accuracy (Ac) with histological verification. The study used 70 excised breast cancer specimens with different morphological structure and molecular status (Luminal A, Luminal B, Her2/Neo+, non-luminal and triple-negative cancer). Our first aim was to formulate convenient criteria of visual assessment of CP-OCT and C-OCE images intended (i) to differentiate tumorous and non-tumorous tissues and (ii) to enable more precise differentiation among different malignant states. We identified such criteria based on the presence of heterogeneities and characteristics of signal attenuation in CP-OCT images, as well as the presence of inclusions/mosaic structures combined with visually feasible assessment of several stiffness grades in C-OCE images. Secondly, we performed a blinded reader study of the Ac of C-OCE versus CP-OCT, for delineation of tumorous versus non-tumorous tissues followed by identification of breast cancer subtypes. For tumor detection, C-OCE showed higher specificity than CP-OCT (97.5% versus 93.3%) and higher Ac (96.0 versus 92.4%). For the first time, the Ac of C-OCE and CP-OCT were evaluated for differentiation between non-invasive and invasive breast cancer (90.4% and 82.5%, respectively). Furthermore, for invasive cancers, the difference between invasive but low-aggressive and highly-aggressive subtypes can be detected. For differentiation between non-tumorous tissue and low-aggressive breast-cancer subtypes, Ac was 95.7% for C-OCE and 88.1% for CP-OCT. For differentiation between non-tumorous tissue and highly-aggressive breast cancers, Ac was found to be 98.3% for C-OCE and 97.2% for CP-OCT. In all cases C-OCE showed better diagnostic parameters independently of the tumor type. These findings confirm the high potential of OCT-based examinations for rapid and accurate diagnostics during breast conservation surgery.

Histological validation of in vivo assessment of cancer tissue inhomogeneity and automated morphological segmentation enabled by Optical Coherence Elastography.

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.

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

Emerging methods of anti-tumor therapies require new approaches to tumor response evaluation, especially enabling label-free diagnostics and in vivo 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 in vivo monitoring of individual tumor response to therapies without taking multiple biopsies.

OCT-elastography-based optical biopsy for breast cancer delineation and express assessment of morphological/molecular subtypes.

Application of compressional optical coherence elastography (OCE) for delineation of tumor and peri-tumoral tissue with simultaneous assessment of morphological/molecular subtypes of breast cancer is reported. The approach is based on the ability of OCE to quantitatively visualize stiffness of studied samples and then to perform a kind of OCE-based biopsy by analyzing elastographic B-scans that have sizes ~several millimeters similarly to bioptates used for "gold-standard" histological examinations. The method relies on identification of several main tissue constituents differing in their stiffness in the OCE scans. Initially the specific stiffness ranges for the analyzed tissue components (adipose tissue, fibrous and hyalinized tumor stroma, lymphocytic infiltrate and agglomerates of tumor cells) are determined via comparison of OCE and morphological/molecular data. Then assessment of non-tumor/tumor regions and tumor subtypes is made based on percentage of pixels with different characteristic stiffness ("stiffness spectrum") in the OCE image, also taking into account spatial localization of different-stiffness regions. Examples of high contrast among benign (or non-invasive) and several subtypes of invasive breast tumors in terms of their stiffness spectra are given.

Raster-scan optoacoustic angiography of blood vessel development in colon cancer models.

Raster-scan optoacoustic angiography at 532 nm wavelength with 50 µm lateral resolution at 2 mm diagnostic depth was used for quantitative characterization of neoangiogenesis in colon cancer models. Two tumor models of human colon adenocarcinoma (HT-29) and murine colon carcinoma (CT26) different in their histology and vascularization were compared. Tumors of both origins showed an inhomogeneous distribution of areas with high and low vascularization. Rapidly growing CT26 tumor demonstrated a higher rate of vessel growth from the periphery to the center. Peculiarities of the vascularity of tumor models revealed by optoacoustic imaging were confirmed by fluorescent microscopy with FITC-dextran and morphological analysis. The obtained results may be important for the investigation of tumor development and for improvement of colon cancer treatment strategies.