Shuhua Yue

Multimodal Nonlinear Optical Microscopy

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed.

Label-Free Analysis of Breast Tissue Polarity by Raman Imaging of Lipid Phase

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger the breast epithelium. A Raman scattering-based platform was used for label-free determination of apical polarity in live breast glandular structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces of an acinus. Raman microspectroscopy subsequently revealed that polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca(2+)-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore, the disruption of apical polarity by a dietary breast cancer risk factor, ω6 fatty acid, could be observed with this method, even when the effect was too moderate to permit a conclusive assessment by the traditional immunostaining method.

Cholesterol Esterification Inhibition Suppresses Prostate Cancer Metastasis by Impairing the Wnt/β-catenin Pathway

Dysregulation of cholesterol is a common characteristic of human cancers including prostate cancer. This study observed an aberrant accumulation of cholesteryl ester in metastatic lesions using Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intracardiac injection mouse models. Gene expression profiling reveals that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. In addition, Wnt/β-catenin, a vital pathway for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. Mol Cancer Res; 16(6); 974–85. ©2018 AACR.

Abstract A06: Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness

Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single-cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism. Citation Format: Hyeon Jeong Lee, Shuhua Yue, Junjie Li, Seung-Young Lee, Tian Shao, Bing Song, Liang Cheng, Timothy A. Masterson, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A06.

Abstract 1893: Spectroscopic imaging unveils altered cholesterol metabolism in prostate cancer .

Metabolic reprogramming allows cancer cells to sustain high proliferative rates and resist cell death. Whereas alterations to glucose and amino acid metabolism have been extensively studied, altered lipid metabolism in cancer is increasingly recognized based on the findings of imbalanced lipid signaling network and dysregulated lipogenic enzymes. Storage of neutral lipids in lipid droplets (LDs), an essential aspect of lipid metabolism, is however long underappreciated until recent advances in LD biology. In human prostate cancer cell lines, neutral lipid accumulation was commonly seen and partially attributed to the up-regulation of fatty acid synthase, a key lipogenic enzyme catalyzing fatty acid biosynthesis and implicated in many human malignancies including prostate cancer. However, because the composition of LDs is not accessible with fluorescence microscopy, the exact role of lipid accumulation in prostate cancer progression remains elusive. By vibration-based spectroscopic imaging of 65 specimens collected from 61 prostate cancer patients and healthy donors, we found for the first time a marked shift from lipofuscin-like autofluorescent granules in normal, benign, and prostatic intraepithelial neoplasia lesions to cholesteryl ester (CE)-rich LDs in high-grade and metastatic prostate cancers. Quantitatively, CE amount was found ∼5 folds higher in high-grade relative to low-grade prostate cancers. Cellular studies demonstrate that the activated PI3K/Akt/mTOR/SREBP pathway accounts for accumulation of CEs via elevated LDL uptake and cholesterol esterification. Blockage of cholesterol esterification with an acetyl-coA cholesterol acyltransferase (ACAT) inhibitor significantly slows the growth and induces apoptosis of prostate cancer both in vitro and in vivo. These findings suggest that CE can be a quantifiable molecular marker for diagnosis of prostate cancer, especially for differentiation between low-grade and high-grade prostate cancers which currently has up to 40% inter-observer discordance. Moreover, targeting the altered cholesterol metabolism opens new opportunities for treating advanced prostate cancer in human patients. Citation Format: Shuhua Yue, Junjie Li, Seung Young Lee, Tian Shao, Bing Song, Liang Cheng, Chang-Deng Hu, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng. Spectroscopic imaging unveils altered cholesterol metabolism in prostate cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2013-1893 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3306: A novel reconstructed metastasis (rMet) model to understand the role of breast cancer stem cells in metastasis

Breast cancer is the most common malignancy in women, with 80% of patients suffering from invasive tumors and metastatic disease. Metastasis is a process where breast cancer cells disseminate from the mammary gland, invade through the extracellular matrix (ECM), intravasate, colonize a distant site such as the bone marrow (BM), and establish growing metastases. Further complicating the metastatic process is the requirement for permissive microenvironment at the secondary site to allow its colonization by the metastatic cells. It has been proposed that ‘cancer stem cells’ (CSCs) with the ability to self-renew, can drive the growth and spread of the tumor. However, the contribution of the breast CSCs (B-CSCs) to metastatic disease is not completely understood. The purpose of this study is to develop a novel 3-dimensional (3-D) tissue culture model to delineate the molecular events responsible for tumor metastasis and to determine whether B-CSCs are responsible for metastatic spread. We developed a ‘reconstructed metastasis’ (rMet) model that mimics the process of breast cancer metastasis to the bone. Using the MCF10A series of isogenic breast cancer progression cell lines, we evaluated the phenotype of metastatic MCF10CA1a cells based on the pattern of expression of epithelial, mesenchymal and B-CSC markers. We used zymography, electron microscopy, and non-linear optical imaging to evaluate ECM remodeling within the rMet system. We observed 3 distinct cell populations emerging in the rMet system: (1) a non-migratory, (2) an invasive, and (3) BM-colonizing, all with distinct phenotypes and genetic signature. A sub-population of non-malignant (MCF10A), pre-malignant (MCF10AneoT), malignant, but not metastatic (MCF10CA1h), and metastatic (MCF10CA1a) cell lines had the capacity to invade BM matrix. However, only a rare population of MCF10CA1a cells, morphologically distinct from their non-migratory counterparts, had the ability to survive and colonize the BM ECM. We found that the BM-colonizing cells display the B-CSC phenotype (CD44+CD24−ESA+ and ALDH+) suggesting their importance in the metastatic spread of breast cancer. The BM colonizing cells expressed a mixture of epithelial and mesenchymal markers, N-cadherin, keratin 18, fibronectin and vimentin, but not E-cadherin or keratin 17. Matrix degradation was observed in the ‘mammary gland’ matrix, but not the BM matrix indicating the possible role of matrix metalloproteases in ECM invasion by the primary tumor, but not in the colonization of a distant site. The rMet model recapitulates the complexity of metastatic disease and hence is a robust system to study bone matrix colonization by breast cancer cells. Identification of B-CSCs in the BM colonizing population signifies their role as a specific population mediating spread and hence emphasizes the requirement of new preventive strategies to target B-CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3306. doi:1538-7445.AM2012-3306

A femtosecond stimulated Raman loss (fSRL) microscope for highly sensitive bond-selective imaging

We demonstrate nonlinear vibrational imaging of isolated Raman bands by detecting femtosecond pulse stimulated Raman loss. Femtosecond pulse excitation produces a stimulated Raman loss signal that is 12 times larger than what picosecond pulse excitation produces. The strong signal allowed real-time, bond-selective imaging of deuterated palmitic acid-d31 inside live cells, and 3D sectioning of fat storage in live C. elegans. With the high peak power provided by femtosecond pulses, this system is highly compatible with other nonlinear optical modalities such as two-photon excited fluorescence. With most of the excitation power contributed by the Stokes beam in the 1.0 - 1.2 μm wavelength range, photodamage of biological samples was not observed.

Abstract A19: Label-free analysis of tissue polarity in live three-dimensional culture of breast epithelium by Raman imaging of lipid phase

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger or protect the breast epithelium. A Raman scattering based platform was used for label-free determination of apical polarity in live breast epithelial structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces in the equatorial plane of an acinus. Raman microspectroscopy subsequently revealed that in polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca2+-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore the disruption of apical polarity by dietary breast cancer risk factor, 6 fatty acid, could be observed significantly with this method, even when the effect was too moderate to permit a conclusive assessment by visually scoring the acinus population for apical location of tight junction marker ZO-1. Analysis of apical polarity markers and cytoskeleton by fluorescence microcopy revealed that the change of lipid ordering upon apical polarity loss corresponded to actual disruption of the apical polarity complex. Our label-free platform will enable rapid identification of environmental factors that either initiate or prevent the very early stage of breast neoplasia. Identifying risk and protective factors will facilitate the design of primary prevention strategies to reduce breast cancer incidence. Furthermore, the use of the membrane lipid phase as readout to assess tissue polarity should be applicable to other types of epithelia where over 90% of cancers originate. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A19.

Abstract 1883: Highly effective screening of breast cancer risk factors by compound Raman analysis of 3D culture of human mammary epithelium

The factors responsible for the transition from a normally differentiated breast epithelium to a neoplastic lesion remain largely undeciphered. Loss of apical polarity is one of the earliest changes observed in tissue architecture in breast neoplasia. However, the microenvironmental factors that control apical polarity are not known. We propose that screening for factors that control apical polarity might provide key answers regarding mechanisms that are involved in very early alterations necessary to permit breast cancer development. In this work, we applied compound Raman microscopy, which combines coherent anti-Stokes Raman scattering (CARS) imaging and confocal Raman microspectroscopy, to simultaneously mapping lipid distribution and analyzing lipid phases of live human mammary epithelium in a high throughput (HTP) 3D culture model that recapitulates polarized mammary acini. Owing to EGTA (ethylene glycol tetraacetic acid) treatment, which significantly induces the loss of apical polarity, we analyzed a total of 40 polarized and 40 nonpolarized mammary acini. The degree of lipid ordering, represented by the ratio between the areas under Raman bands at 2885 cm-1 (A2885) and 2850 cm-1 (A2850), A2885/A2850 (R), was found significantly higher in apical membrane (ApM) relative to basal membrane (BaM) for polarized mammary acini, but significantly lower for nonpolarized mammary acini by using student9s t-test. According to this result, a ratio ApM_R / BaM_R was defined to represent the relative degree of lipid ordering of ApM compared with BaM. We then found that ApM_R / BaM_R successfully distinguished polarized and nonpolarized mammary acini at a cut-off of 1 with high sensitivity and specificity. We, for the first time, provide direct evidence of polarized sorting of lipids in live human mammary epithelium. Most importantly, the same mammary acinus undergoing treatment could be monitored over a period of time, which revealed the dynamic changes of apical polarity without fixation and staining. Thus, compound Raman microscopy could eliminate the uncertainty caused by acinus-to-acinus variability and become an effective and accurate screening method of breast cancer risk factors that act on tissue polarity. Among the studies that prevail, there is enough evidence to support a role of nutrition and diet in breast cancer incidence. Our screening platform should assist in identifying certain types of fatty acids (FAs) and other dietary factors that initiate the very early stage of breast neoplasia as well as compounds that might help maintain breast tissue integrity and thus protect against breast neoplasia. The ultimate list of risk and protective nutritional factors could promote precise mechanistic studies on how these factors affect apical polarity, and set up a solid basis for the design of better prevention and therapeutic strategies to reduce breast cancer incidence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1883.