Chao Zheng

Raman spectra exploring breast tissues: Comparison of principal component analysis and support vector machine‐recursive feature elimination

PURPOSE Raman spectroscopy was explored to diagnose normal, benign, and malignant human breast tissues based on principal component analysis (PCA) and support vector machine-recursive feature elimination (SVM-RFE), and SVM-RFE results were compared with PCA. METHODS 1800 Raman spectra were acquired from fresh samples of human breast tissues (normal, fibroadenoma, adenosis, galactoma, and invasive ductal carcinoma) from 168 patients. After set up the SVM-RFE and PCA models, Mahalanobis distance, spectral residuals, sensitivity, specificity, and Matthews correlation coefficient (MCC) were used as the discriminating criteria for evaluating these two methods. RESULTS The comparison shows that SVM-RFE based on the selection of characteristic peaks better reflects the nature of biopsy and it produces better discrimination, sensitivity, specificity, and MCC for normal (1, 1, 1), malignant (0.93, 0.97, 0.91), and benign (0.95, 0.97, 0.91) breast tissues. CONCLUSIONS The Raman spectroscopy combined with SVM-RFE opens great future in the clinical applications of mammary disease diagnosis.PURPOSE Raman spectroscopy was explored to diagnose normal, benign, and malignant human breast tissues based on principal component analysis (PCA) and support vector machine-recursive feature elimination (SVM-RFE), and SVM-RFE results were compared with PCA. METHODS 1800 Raman spectra were acquired from fresh samples of human breast tissues (normal, fibroadenoma, adenosis, galactoma, and invasive ductal carcinoma) from 168 patients. After set up the SVM-RFE and PCA models, Mahalanobis distance, spectral residuals, sensitivity, specificity, and Matthews correlation coefficient (MCC) were used as the discriminating criteria for evaluating these two methods. RESULTS The comparison shows that SVM-RFE based on the selection of characteristic peaks better reflects the nature of biopsy and it produces better discrimination, sensitivity, specificity, and MCC for normal (1, 1, 1), malignant (0.93, 0.97, 0.91), and benign (0.95, 0.97, 0.91) breast tissues. CONCLUSIONS The Raman spectroscopy combined with SVM-RFE opens great future in the clinical applications of mammary disease diagnosis.

The use of Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy for human breast cancer detection

AbstractThis study uses the powerful fingerprint features of Raman spectroscopy to distinguish different types of breast tissues including normal breast tissues (NB), fibroadenoma (FD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Thin frozen tissue sections of fresh breast tissues were measured by Raman spectroscopy. Due to the inherent low sensitivity of Raman spectra, Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized to provide supplementary and more informative spectral features. A total of 619 Raman spectra were acquired and compared to 654 SHINERS spectra. The maximum enhancement effect of distinct and specific bands was characterized for different tissue types. When applying the new criteria, excellent separation of FD, DCIS, and IDC was obtained for all tissue types. Most importantly, we were able to distinguish ADH from DCIS. Although only a preliminary distinction was characterized between ADH and NB, the results provided a good foundation of criteria to further discriminate ADH from NB and shed more light toward a better understanding of the mechanism of ADH formation. This is the first report to detect the premalignant (ADH and DCIS) breast tissue frozen sections and also the first report exploiting SHINERS to detect and distinguish breast tissues. The results presented in this study show that SHINERS can be applied to accurately and efficiently identify breast lesions. Further, the spectra can be acquired in a minimally invasive procedure and analyzed rapidly facilitating early and accurate diagnosis in vivo/in situ. FigureHuman breast cancer detection with Au@SiO2 SHINERS

Label-Free Raman Spectroscopy Detects Stromal Adaptations in Premetastatic Lungs Primed by Breast Cancer.

Recent advances in animal modeling, imaging technology, and functional genomics have permitted precise molecular observations of the metastatic process. However, a comprehensive understanding of the premetastatic niche remains elusive, owing to the limited tools that can map subtle differences in molecular mediators in organ-specific microenvironments. Here, we report the ability to detect premetastatic changes in the lung microenvironment, in response to primary breast tumors, using a combination of metastatic mouse models, Raman spectroscopy, and multivariate analysis of consistent patterns in molecular expression. We used tdTomato fluorescent protein expressing MDA-MB-231 and MCF-7 cells of high and low metastatic potential, respectively, to grow orthotopic xenografts in athymic nude mice and allow spontaneous dissemination from the primary mammary fat pad tumor. Label-free Raman spectroscopic mapping was used to record the molecular content of premetastatic lungs. These measurements show reliable distinctions in vibrational features, characteristic of the collageneous stroma and its cross-linkers as well as proteoglycans, which uniquely identify the metastatic potential of the primary tumor by recapitulating the compositional changes in the lungs. Consistent with histological assessment and gene expression analysis, our study suggests that remodeling of the extracellular matrix components may present promising markers for objective recognition of the premetastatic niche, independent of conventional clinical information. Cancer Res; 77(2); 247-56. ©2016 AACR.

Exploring type II microcalcifications in benign and premalignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)

The characteristics of type II microcalcifications in fibroadenoma (FB), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) breast tissues has been analyzed by the fingerprint features of Raman spectroscopy. Fresh breast tissues were first handled to frozen sections and then they were measured by normal Raman spectroscopy. Due to inherently low sensitivity of Raman scattering, Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized. A total number of 71 Raman spectra and 70 SHINERS spectra were obtained from the microcalcifications in benign and premalignant breast tissues. Principal component analysis (PCA) was used to distinguish the type II microcalcifications between these tissues. This is the first time to detect type II microcalcifications in premalignant (ADH and DCIS) breast tissue frozen sections, and also the first time SHINERS has been utilized for breast cancer detection. Conclusions demonstrated in this paper confirm that SHINERS has great potentials to be applied to the identification of breast lesions as an auxiliary method to mammography in the early diagnosis of breast cancer.

COX-2 silencing enhances tamoxifen antitumor activity in breast cancer in vivo and in vitro

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. However, extended TAM administration for breast cancer induces increased VEGF levels in patients, promoting new blood vessel formation and thereby limiting its efficacy and highlighting the need for improved therapeutic strategies. Cyclooxygenase-2 (COX-2) silencing via a replication-incompetent lentivirus (LV-COX-2) induce cancer apoptosis and suppresses VEGF gene expression. In this study, the effect of LV-COX-2 infection, either alone or in combination with TAM, was analyzed in a breast cell lines for suppressing VEGF expression and simultaneously reducing doses of TAM. Cell proliferation, apoptosis, angiogenesis, metastasis, cell cycle distribution, an receptor signaling were determined after LV-COX-2 combination with TAM treatment. In addition, tumor growth ability in nude mice was detected to define the combination treatment effect in tumorigenesis in vivo. It is found that LV-COX-2 combination with TAM treatment in breast cancer cell significantly suppressed the proliferation and metastasis, and induced tumor apoptosis in vitro, and tumor growth also was suppressed in vivo. In addition, we also found that LV-COX-2 combination with TAM treatment could inhibit angiogenesis and VEGF expression. Taken together, our experimental results indicate that LV-COX-2 combination with TAM has promising outcome in anti-metastatic and apoptotic studies. Furthermore, these results showed that LV-COX-2 combination with TAM is a potential drug candidate for treatment of breast tumors expressing high levels of VEGF.

The Clinical Application of Raman Spectroscopy for Breast Cancer Detection

Raman spectroscopy has been widely used as an important clinical tool for real-time in vivo cancer diagnosis. Raman information can be obtained from whole organisms and tissues, at the cellular level and at the biomolecular level. The aim of this paper is to review the newest developments of Raman spectroscopy in the field of breast cancer diagnosis and treatment. Raman spectroscopy can distinguish malignant tissues from noncancerous/normal tissues and can assess tumor margins or sentinel lymph nodes during an operation. At the cellular level, Raman spectra can be used to monitor the intracellular processes occurring in blood circulation. At the biomolecular level, surface-enhanced Raman spectroscopy techniques may help detect the biomarker on the tumor surface as well as evaluate the efficacy of anticancer drugs. Furthermore, Raman images reveal an inhomogeneous distribution of different compounds, especially proteins, lipids, microcalcifications, and their metabolic products, in cancerous breast tissues. Information about these compounds may further our understanding of the mechanisms of breast cancer.

Abstract P2-05-15: Exploring type II microcalcifications in benign, premalignant and malignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)

Background: Type II microcalcifications, most often seen in proliferative lesions, including both benign and malignant pathologies. But whether the emergence of type II microcalcifications associated with cell canceration is still not clear now. Raman spectroscopy is a powerful, non-invasive analytical tool which can provide detailed and meaningful information about biochemical composition of tissues at molecular level. Our aim was to find the differences and relationships of type II microcalcifications between fibroadenoma, ADH tissues, and DCIS, IDC in breast based on their various biochemical characteristics by Raman spectroscopy. Methods: The frozen sections were collected from 15 patients (all female; ages 25-57) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20∼-25° and two contiguous sections (6 µm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis and found the microcalcification locations by three expert breast pathologists; the same position of the other section was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the HE San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-15.

Mapping the genetic basis of breast microcalcifications and their role in metastasis

Breast cancer screening and early stage diagnosis is typically performed by X-ray mammography, which detects microcalcifications. Despite being one of the most reliable features of nonpalpable breast cancer, the processes by which these microcalcifications form are understudied and largely unknown. In the current work, we have investigated the genetic drivers for the formation of microcalcifications in breast cancer cell lines, and have investigated their involvement in disease progression. We have shown that stable silencing of the Osteopontin (OPN) gene decreased the formation of hydroxyapatite in MDA-MB-231 breast cancer cells in response to osteogenic cocktail. In addition, OPN silencing reduced breast cancer cell migration. Furthermore, breast cancer cells that had spontaneously metastasized to the lungs in a mouse model of breast cancer had largely elevated OPN levels, while circulating tumor cells in the same mouse model contained intermediately increased OPN levels as compared to parental cells. The observed dual roles of the OPN gene reveal the existence of a direct relationship between calcium deposition and the ability of breast cancer cells to metastasize to distant organs, mediated by common genetic factors.

Abstract P6-01-18: Early detection of development of a pre-metastatic niche in lungs in response to primary breast tumor using Raman spectroscopy

Background: An alarmingly large proportion of cancer-related deaths result from metastatic cancers. Development of quick, reliable and non-invasive or minimally invasive approaches to objectively assess the secondary tissues (potentially in vivo) will be instrumental in substantially reducing the cancer burden due to metastasis, which accounts for majority of cancer related mortality. In our study we report the utilization of Raman spectroscopy and chemometric techniques in identifying formation of pre-metastatic niche in lungs prior to observing morphological changes. Methods: Six-week-old female athymic nu/nu mice (NCI, MD) were implanted with 2x106 cells of human breast cancer cell lines - MDA-MB-231 (n=3), and MCF-7 (n=3) in their fourth right mammary fat pad orthotopically. And control mice (n=3) without tumor cell implantation were also employed. The primary tumor size was monitored and the mice were sacrificed within 8-12 weeks of cell implantation when the primary tumors volume grew to 500-600 mm3. Control mice were also sacrificed in this timeframe. The freshly excised lungs of the mice were cleaned in PBS and utilized for obtaining Raman spectra (830 nm, thermoelectrically cooled CCD). Each tissue was collected from multiple points. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) were employed as discriminating algorithm. Following spectral acquisition, the tissues were fixed in 10% formalin, embedded in paraffin, and then HE staining, and Masson9s trichrome staining for collagen. Collagen quantification of Masson9s trichrome stained slides was achieved using MATLAB (Mathworks, MA). The Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine approved the protocol of the study. Results: 900 Raman spectra each acquired from the lungs of the control mice and mice bearing MCF-7 and MDA-MB-231 tumor xenografts were assigned class labels - 9Control9, 9MCL9 and 9MDL9 respectively for further analysis. Select principal components from those obtained by subjecting all the chosen 900 spectra to PCA clearly evident that the differences in the Raman spectra belonging to tissues being primed by derivatives of different primary cells are quite pronounced. The average correct rates of PLS-DA prediction of 90.1%, 97.7% and 78.4% were obtained for the spectra belonging to the classes - Control, MCL and MDL respectively. The HE images are negative for any signs of cancerous lesions. Masson9s trichrome staining results show that the metastatic potential of the cell lines responsible for the primary tumor is positively correlated with the collagen density in the pre-metastatic niche, the MDL shows the highest collagen density (P Conclusion: The current study introduces Raman spectroscopy in conjunction with chemometric techniques as are liable and minimally invasive tool for diagnosis of metastatic cancers significantly early in the metastatic cascade, and also opens a new route for early targeting of cancer metastasis and its associated burden. Citation Format: Zheng C, Rizwan A, Paidi SK, Yu Z, Barman I, Glunde K. Early detection of development of a pre-metastatic niche in lungs in response to primary breast tumor using Raman spectroscopy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-18.

Probing nanoscopic cell surface areas for rapid and label-free plasmon enhanced Raman detection

Surface enhanced Raman spectroscopy (SERS) is an emerging nanoscale tool for small quality sample probing. Its highly localized detection range makes it suits for cell membrane composition research. In this research, we combined SERS with nanopillar array to identify aggressive prostate cancer cell surface markers. Inherent signal fluctuations emanating from cell lines with differential biomarker expression highlight the potential of using such a nanopillar-based platform for probing the chemical and mechanical phenotype and pave the way for evolution of a direct and label free assessment of cancer membrane biomarkers.