Detection of pathological response of axillary lymph node metastasis after neoadjuvant chemotherapy in breast cancer using multiphoton microscopy

Neoadjuvant treatment is often considered in breast cancer patients with axillary lymph node involvement, but most of patients do not have a pathologic complete response to therapy. The detection of residual nodal disease has a significant impact on adjuvant therapy recommendations which may improve survival. Here, we investigate whether multiphoton microscopy (MPM) could identify the pathological changes of axillary lymphatic metastasis after neoadjuvant chemotherapy in breast cancer. And furthermore, we find that there are obvious differences in seven collagen morphological features between normal node and residual axillary disease by combining with a semi-automatic image processing method, and also find that there are significant differences in four collagen features between the effective and no-response treatment groups. These research results indicate that MPM may help estimate axillary treatment response in the neoadjuvant setting and thereby tailor more appropriate and personalized adjuvant treatments for breast cancer patients.     

In vivo detection of tumor boundary using ultrahigh‐resolution optical coherence angiography and fluorescence imaging

Accurate detection of early tumor margin is of great preclinical and clinical implications for predicting the survival rate of subjects and assessing the response of tumor microenvironment to chemotherapy or radiation therapy. Here, we report a multimodality optical imaging study on in vivo detection of tumor boundary by analyzing neoangiogenesis of tumor microenvironment (microangiography), microcirculatory blood flow (optical Doppler tomography) and tumor proliferation (green fluorescent protein [GFP] fluorescence). Microangiography demonstrates superior sensitivity (77.7 ± 6.4%) and specificity (98.2 ± 1.7%) over other imaging technologies (eg, optical coherence tomography) for tumor margin detection. Additionally, we report longitudinal in vivo imaging of tumor progression and show that the abrupt tumor cell proliferation did not occur until local capillary density and cerebral blood flow reached their peak approximately 2 weeks after tumor implantation. The unique capability of longitudinal multimodality imaging of tumor angiogenesis may provide new insights in tumor biology and in vivo assessment of the treatment effects on anti‐angiogenesis therapy for brain cancer.     

Biophotonic technologies for assessment of breast tumor surgical margins—A review

Breast conserving surgery (BCS) offering similar surgical outcomes as mastectomy while retaining breast cosmesis is becoming increasingly popular for the management of early stage breast cancers. However, its association with reoperation rates of 20% to 40% following incomplete tumor removal warrants the need for a fast and accurate intraoperative surgical margin assessment tool that offers cellular, structural and molecular information of the whole specimen surface to a clinically relevant depth. Biophotonic technologies are evolving to qualify as such an intraoperative tool for clinical assessment of breast cancer surgical margins at the microscopic and macroscopic scale. Herein, we review the current research in the application of biophotonic technologies such as photoacoustic imaging, Raman spectroscopy, multimodal multiphoton imaging, diffuse optical imaging and fluorescence imaging using medically approved dyes for breast cancer detection and/or tumor subtype differentiation toward intraoperative assessment of surgical margins in BCS specimens, and possible challenges in their route to clinical translation.     

Wide‐field monitoring and real‐time local recording of microvascular networks on small animals with a dual‐raster‐scanned photoacoustic microscope

Photoacoustic microscopy (PAM) provides a new method for the imaging of small‐animals with high‐contrast and deep‐penetration. However, the established PAM systems have suffered from a limited field‐of‐view or imaging speed, which are difficult to both monitor wide‐field activity of organ and record real‐time change of local tissue. Here, we reported a dual‐raster‐scanned photoacoustic microscope (DRS‐PAM) that integrates a two‐dimensional motorized translation stage for large field‐of‐view imaging and a two‐axis fast galvanometer scanner for real‐time imaging. The DRS‐PAM provides a flexible transition from wide‐field monitoring the vasculature of organs to real‐time imaging of local dynamics. To test the performance of DRS‐PAM, clear characterization of angiogenesis and functional detail was illustrated, hemodynamic activities of vasculature in cerebral cortex of a mouse were investigated. Furthermore, response of tumor to treatment were successfully monitored during treatment. The experimental results demonstrate the DRS‐PAM holds the great potential for biomedical research of basic biology.     

Distinguishing metastatic triple‐negative breast cancer from nonmetastatic breast cancer using second harmonic generation imaging and resonance Raman spectroscopy

Triple‐negative breast cancer (TNBC) is an aggressive subset of breast cancer that is more common in African‐American and Hispanic women. Early detection followed by intensive treatment is critical to improving poor survival rates. The current standard to diagnose TNBC from histopathology of biopsy samples is invasive and time‐consuming. Imaging methods such as mammography and magnetic resonance (MR) imaging, while covering the entire breast, lack the spatial resolution and specificity to capture the molecular features that identify TNBC. Two nonlinear optical modalities of second harmonic generation (SHG) imaging of collagen, and resonance Raman spectroscopy (RRS) potentially offer novel rapid, label‐free detection of molecular and morphological features that characterize cancerous breast tissue at subcellular resolution. In this study, we first applied MR methods to measure the whole‐tumor characteristics of metastatic TNBC (4T1) and nonmetastatic estrogen receptor positive breast cancer (67NR) models, including tumor lactate concentration and vascularity. Subsequently, we employed for the first time in vivo SHG imaging of collagen and ex vivo RRS of biomolecules to detect different microenvironmental features of these two tumor models. We achieved high sensitivity and accuracy for discrimination between these two cancer types by quantitative morphometric analysis and nonnegative matrix factorization along with support vector machine. Our study proposes a new method to combine SHG and RRS together as a promising novel photonic and optical method for early detection of TNBC.     

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.     

Evaluation of changes in serum protein profiles during neoadjuvant chemotherapy in HER2‐positive breast cancer using an LC‐MALDI‐TOF/MS procedure

Comparison of protein profiles of sera acquired before and after preoperative chemotherapy for breast cancer may reveal tumor markers that could be used to monitor tumor response. In this study, we analyzed pre‐ and post‐chemotherapy protein profiles of sera from 39 HER2‐postive breast cancer patients (n=78 samples) who received 6 months of preoperative chemotherapy using LC‐MALDI‐TOF/MS technology. We detected qualitative and quantitative differences in pair‐wise comparison of pre‐ and post chemotherapy samples that were different in patients who achieved pathological complete response (pCR, n=21) compared with those with residual disease (n=18). We identified 2329 and 3152 peaks as differentially expressed in the pre‐chemotherapy samples of the responders and non‐responders. Comparison of matching pre‐ and post‐chemotherapy samples identified 34 (32 decreased, two increased) and 304 peaks (157 decreased, 147 increased) that significantly changed (p<0.01, false discovery rate ≤20%) after treatment in responders and non‐responders, respectively. The top 11 most significantly altered peptide peaks with the greatest change in intensity were positively identified. These corresponded to eight proteins including α‐2‐macroglobulin, complement 3, hemopexin, and serum amyloid P in the responder group and chains C and A of apolipoprotein A‐I, hemopexin precursor, complement C, and amyloid P component in the non‐responding groups. All proteins decreased after therapy, except chain C apolipoprotein A and hemopexin precursor that increased. These results suggest that changes in serum protein levels occur in response to chemotherapy and these changes partly appear different in patients who are highly sensitive to chemotherapy compared with those with lesser response.     

In vivo longitudinal imaging of RNA interference‐induced endocrine therapy resistance in breast cancer

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor‐positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence‐labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)‐regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox‐induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor‐bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen‐treated control group. Longitudinal imaging was performed by a home‐built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper‐measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e‐7) correlated with the tumor size progression for the mice with shRNA‐induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.      

Label-free detection of invasive micropapillary carcinoma of the breast using multiphoton microscopy

Invasive micropapillary carcinoma of the breast (IMPC) is a rare form of breast cancer with unique histological features, and is associated with high axillary lymph node metastasis and poor clinical prognosis. Thus, IMPC should be diagnosed in time to improve the treatment and management of patients. In this study, multiphoton microscopy (MPM) is used to label-free visualize the morphological features of IMPC. Our results demonstrate that MPM images are well in agreement with hematoxylin and eosin staining and epithelial membrane antigen staining, indicating MPM is comparable to traditional histological analysis in identifying the tissue structure and cell morphology. Statistical analysis shows significant differences in the circumference and area of the glandular lumen and cancer nest between the different IMPC cell clusters with complete glandular lumen morphology, and also shows difference in collagen length, width, and orientation, indicating the invasive ability of different morphologies of IMPC may be different.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.     

Integrated photoacoustic and hyperspectral dual‐modality microscopy for co‐imaging of melanoma and cutaneous squamous cell carcinoma in vivo

Skin carcinoma such as melanoma (MM) and cutaneous squamous cell carcinoma (cSCC) are considered as the highest mortality and the most aggressive skin cancers in dermatology. In view that early diagnosis and treatment can greatly improve the survival rate and life quality of the patients, developing noninvasive and effective evaluation methods is of great significance for the detection and identification of early stage cutaneous cancers. In this article, we propose a hybrid photoacoustic and hyperspectral dual‐modality microscopy to evaluate and differentiate skin carcinoma by structural and multiphysiological parameters. The proposed system's imaging abilities are verified by mimic phantoms and normal mice experiments. Furthermore, in vivo characterization and evaluation results of MM and cSCC mice are obtained successfully, which prove this novel method could be used as a reliable and useful method for skin cancer detection in early stages.     

Three‐dimensional mapping of the attenuation coefficient in optical coherence tomography to enhance breast tissue microarchitecture contrast

Effective intraoperative tumor margin assessment is needed to reduce re‐excision rates in breast‐conserving surgery (BCS). Mapping the attenuation coefficient in optical coherence tomography (OCT) throughout a sample to create an image (attenuation imaging) is one promising approach. For the first time, three‐dimensional OCT attenuation imaging of human breast tissue microarchitecture using a wide‐field (up to ~45 × 45 × 3.5 mm) imaging system is demonstrated. Representative results from three mastectomy and one BCS specimen (from 31 specimens) are presented with co‐registered postoperative histology. Attenuation imaging is shown to provide substantially improved contrast over OCT, delineating nuanced features within tumors (including necrosis and variations in tumor cell density and growth patterns) and benign features (such as sclerosing adenosis). Additionally, quantitative micro‐elastography (QME) images presented alongside OCT and attenuation images show that these techniques provide complementary contrast, suggesting that multimodal imaging could increase tissue identification accuracy and potentially improve tumor margin assessment.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Dual-modality monitoring of tumor response to cyclophosphamide therapy in mice with bioluminescence imaging and small-animal positron emission tomography

The purpose of this study was to noninvasively monitor the therapeutic efficacy of cyclophosphamide (CTX) in a mouse model by dual-modality molecular imaging: positron emission tomography (PET) and bioluminescence imaging (BLI). Firefly luciferase (fLuc) transfected HCC-LM3-fLuc human hepatocellular carcinoma cells were injected subcutaneously into BALB/c nude mice to establish the experimental tumor model. Two groups of HCC-LM3-fLuc tumor-bearing mice (n = 7 per group) were treated with saline or CTX (100 mg/kg on days 0, 2, 5, and 7). BLI and (18)F-fluorodeoxyglucose ((18)F-FDG) PET scans were done to evaluate the treatment efficacy. CTX induced a 25.25 ± 13.13% and 35.91 ± 25.85% tumor growth inhibition rate on days 9 and 12 posttreatment, respectively, as determined by BLI. A good linear correlation was found between the tumor sizes measured by caliper and the BLI signals determined by optical imaging (R(2) = .9216). (18)F-FDG imaging revealed a significant uptake reduction in the tumors of the CTX-treated group compared to that in the saline control group (5.30 ± 1.97 vs 3.00 ± 2.11% ID/g) on day 16 after CTX treatment. Dual-modality molecular imaging using BLI and small-animal PET can play important roles in the process of chemotherapy and will provide noninvasive and reliable monitoring of the therapeutic response.     

Unique spectral signature of human cutaneous squamous cell carcinoma by photoacoustic imaging

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer with metastatic potential. To reduce reoperations due to nonradical excision, there is a need to develop a technique for identification of tumor margins preoperatively. Photoacoustic (PA) imaging is a novel imaging technology that combines the strengths of laser optics and ultrasound. Our aim was to determine the spectral signature of cSCC using PA imaging and to use this signature to visualize tumor architecture and borders. Two‐dimensional PA images of 33 cSCCs and surrounding healthy skin were acquired ex vivo, using 59 excitation wavelengths from 680 to 970 nm. The spectral response of the cSCCs was compared to healthy tissue, and the difference was found to be greatest at wavelengths in the range 765 to 960 nm (P < .05). Three‐dimensional PA images were constructed from spectra obtained in the y‐z plane using a linear stepper motor moving along the x‐plane. Spectral unmixing was then performed which provided a clear three‐dimensional view of the distribution of tumor masses and their borders.     

彩色多普勒超声对乳腺癌新辅助化疗疗效的评价研究

目的:探讨彩色多普勒超声(CDFI)对乳腺癌新辅助化疗(NCT)疗效评价的临床应用价值。方法:选取2013年1月~2013年12月在我院接受新辅助化疗后行手术治疗的女性乳腺癌患者55例,以病理学评价为金标准,化疗后根据化疗效果分为有效组和无效组,利用CDFI观察患者NCT前后病灶超声指标、病灶内血流分级及阻力指数(RI)值变化。结果:55例患者中,临床触诊疗效评价符合率为36.4%,敏感度为60.7%;CDFI评价符合率为70.9%,敏感度为85.7%。CDFI检查显示,乳腺癌NCT后原发肿瘤病灶大小显著缩小,边界多清晰可见,内部回声及后方回声倾向正常。有效组NCT前后病灶内的血流类型和RI值变化有统计学意义(P0.05),无效组NCT前后病灶内的血流类型和RI值无明显变化(P0.05)。结论:CDFI技术可对乳腺癌NCT前后病灶大小及病灶内部血流动力学变化提供客观参数,是评价乳腺癌NCT疗效的有效方法。     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

Superpixel Raman spectroscopy for rapid skin cancer margin assessment

Spontaneous Raman micro‐spectroscopy has been demonstrated great potential in delineating tumor margins; however, it is limited by slow acquisition speed. We describe a superpixel acquisition approach that can expedite acquisition between ~×100 and ×10 000, as compared to point‐by‐point scanning by trading off spatial resolution. We present the first demonstration of superpixel acquisition on rapid discrimination of basal cell carcinoma tumor from eight patients undergoing Mohs micrographic surgery. Results have been demonstrated high discriminant power for tumor vs normal skin based on the biochemical differences between nucleus, collagen, keratin and ceramide. We further perform raster‐scanned superpixel Raman imaging on positive and negative margin samples. Our results indicate superpixel acquisition can facilitate the use of Raman microspectroscopy as a rapid and specific tool for tumor margin assessment.     

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high‐speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video‐rate cellular imaging. Moxifloxacin‐based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery‐guiding method for tumor removal.      

Inside Cover: Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

A type of compact and cost‐effective light‐sheet imaging device, termed sub‐voxel‐resolving light‐sheet add‐on module (SLAM), is developed to cooperate with conventional 2D epifluorescence microscope, allowing high‐contrast, resolution‐improved 3D imaging of various biological samples at high throughput. Further details can be found in the article by Fang Zhao, Yicong Yang, Yi Li, et al. ( e201960243 ).