Strategies for High-Resolution Imaging of Epithelial Ovarian Cancer by Laparoscopic Nonlinear Microscopy

Ovarian cancer remains the most frequently lethal of the gynecologic cancers owing to the late detection of this disease. Here, by using human specimens and three mouse models of ovarian cancer, we tested the feasibility of nonlinear imaging approaches, the multiphoton microscopy (MPM) and second harmonic generation (SHG) to serve as complementary tools for ovarian cancer diagnosis. We demonstrate that MPM/SHG of intrinsic tissue emissions allows visualization of unfixed, unsectioned, and unstained tissues at a resolution comparable to that of routinely processed histologic sections. In addition to permitting discrimination between normal and neoplastic tissues according to pathological criteria, the method facilitates morphometric assessment of specimens and detection of very early cellular changes in the ovarian surface epithelium. A red shift in cellular intrinsic fluorescence and collagen structural alterations have been identified as additional cancer-associated changes that are indiscernible by conventional pathologic techniques. Importantly, the feasibility of in vivo laparoscopic MPM/SHG is demonstrated by using a “stick” objective lens. Intravital detection of neoplastic lesions has been further facilitated by low-magnification identification of an indicator for cathepsin activity followed by MPM laparoscopic imaging. Taken together, these results demonstrate that MPM may be translatable to clinical settings as an endoscopic approach suitable for high-resolution optical biopsies as well as a pathology tool for rapid initial assessment of ovarian cancer samples.     

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression

High levels of peripheral-type benzodiazepine receptor (PBR), the alternative-binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non-tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon-rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Integration of Molecular Profiling and Chemical Imaging to Elucidate Fibroblast-Microenvironment Impact on Cancer Cell Phenotype and Endocrine Resistance in Breast Cancer

The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.     

Expression of Peripheral Benzodiazepine Receptor (PBR) in Human Tumors: Relationship to Breast,Colorectal, and Prostate Tumor Progression

Abstract

High levels of peripheral‐type benzodiazepine receptor (PBR), the alternative‐binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non‐tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon‐rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Specific expression of human intelectin-1 in malignant pleural mesothelioma and gastrointestinal goblet cells

Malignant pleural mesothelioma (MPM) is a fatal tumor. It is often hard to discriminate MPM from metastatic tumors of other types because currently, there are no reliable immunopathological markers for MPM. MPM is differentially diagnosed by some immunohistochemical tests on pathology specimens. In the present study, we investigated the expression of intelectin-1, a new mesothelioma marker, in normal tissues in the whole body and in many cancers, including MPM, by immunohistochemical analysis. We found that in normal tissues, human intelectin-1 was mainly secreted from gastrointestinal goblet cells along with mucus into the intestinal lumen, and it was also expressed, to a lesser extent, in mesothelial cells and urinary epithelial cells. Eighty-eight percent of epithelioid-type MPMs expressed intelectin-1, whereas sarcomatoid-type MPMs, biphasic MPMs, and poorly differentiated MPMs were rarely positive for intelectin-1. Intelectin-1 was not expressed in other cancers, except in mucus-producing adenocarcinoma. These results suggest that intelectin-1 is a better marker for epithelioid-type MPM than other mesothelioma markers because of its specificity and the simplicity of pathological assessment. Pleural intelectin-1 could be a useful diagnostic marker for MPM with applications in histopathological identification of MPM.     

Label-free characterization of cancer-activated fibroblasts using infrared spectroscopic imaging

Glandular tumors arising in epithelial cells comprise the majority of solid human cancers. Glands are supported by stroma, which is activated in the proximity of a tumor. Activated stroma is often characterized by the molecular expression of α-smooth muscle actin (α-SMA) within fibroblasts. However, the precise spatial and temporal evolution of chemical changes in fibroblasts upon epithelial tumor signaling is poorly understood. Here we report a label-free method to characterize fibroblast changes by using Fourier transform infrared spectroscopic imaging and comparing spectra with α-SMA expression in primary normal human fibroblasts. We recorded the fibroblast activation process by spectroscopic imaging using increasingly tissue-like conditions: 1), stimulation with the growth factor TGFβ1; 2), coculture with MCF-7 human breast cancerous epithelial cells in Transwell coculture; and 3), coculture with MCF-7 in three-dimensional cell culture. Finally, we compared the spectral signatures of stromal transformation with normal and malignant human breast tissue biopsies. The results indicate that this approach reveals temporally complex spectral changes and thus provides a richer assessment than simple molecular imaging based on α-SMA expression. Some changes are conserved across culture conditions and in human tissue, providing a label-free method to monitor stromal transformations.     

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.     

乳腺癌CXCR1的表达特征及其与新辅助化疗疗效的相关性

目的:研究趋化因子受体CXCR1在乳腺癌组织中的表达特征,并探讨新辅助化疗前后其表达变化与化疗疗效的关系。方法:选取20例正常乳腺组织,20例乳腺纤维腺瘤,104例乳腺癌标本,免疫组化染色后统计每例标本CXCR1的阳性表达积分,分析不同乳腺疾病CXCR1的表达差异。统计新辅助化疗前后乳腺癌标本CXCR1的阳性表达积分的变化,分析其与化疗病理反应的关系。结果:CXCR1在正常乳腺组织、乳腺纤维腺瘤中低表达,在乳腺癌组织上高表达;其表达与患者的年龄、原发肿瘤的大小无关(P0.05),与病理分期、癌细胞的分化程度、淋巴结转移个数、激素受体状态及Her2表达情况相关,P0.05,有统计学意义。新辅助化疗后,癌组织中CXCR1的表达下降,下降幅度越大,其化疗疗效越好。结论:CXCR1的检测对于乳腺疾病的良恶性判断有指导意义,可辅助判断乳腺癌的恶性程度、侵袭性以及预后,CXCR1表达的下降与化疗疗效相关,抑制其表达可能可以提高化疗疗效。     

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.     

Functional heterogeneity of breast fibroblasts is defined by a prostaglandin secretory phenotype that promotes expansion of cancer-stem like cells

Fibroblasts are important in orchestrating various functions necessary for maintaining normal tissue homeostasis as well as promoting malignant tumor growth. Significant evidence indicates that fibroblasts are functionally heterogeneous with respect to their ability to promote tumor growth, but markers that can be used to distinguish growth promoting from growth suppressing fibroblasts remain ill-defined. Here we show that human breast fibroblasts are functionally heterogeneous with respect to tumor-promoting activity regardless of whether they were isolated from normal or cancerous breast tissues. Rather than significant differences in fibroblast marker expression, we show that fibroblasts secreting abundant levels of prostaglandin (PGE2), when isolated from either reduction mammoplasty or carcinoma tissues, were both capable of enhancing tumor growth in vivo and could increase the number of cancer stem-like cells. PGE2 further enhanced the tumor promoting properties of fibroblasts by increasing secretion of IL-6, which was necessary, but not sufficient, for expansion of breast cancer stem-like cells. These findings identify a population of fibroblasts which both produce and respond to PGE2, and that are functionally distinct from other fibroblasts. Identifying markers of these cells could allow for the targeted ablation of tumor-promoting and inflammatory fibroblasts in human breast cancers.     

Promising techniques for breast cancer detection,diagnosis, and staging using non-ionizing radiation imaging techniques

Traditional imaging for the diagnosis and staging of breast cancer has relied on the tissue morphology of cancers in the background of normal patterns of fibroglandular breast tissue. X-ray mammography and ultrasound have been the primary modalities for the diagnosis and the work-up of breast cancer. New modalities have been validated including magnetic resonance imaging (MRI) and positron emission tomography (PET). New pulse sequences in MRI combined with contrast enhancement kinetic perfusion curves have greatly enhanced detection of mammographically occult cancers. New modalities on the horizon include optical imaging, exploiting again the differential perfusion properties of cancers in a background of normal glandular tissue. Even more specificity can be ach eved with the addition of ductal or intravenous introduction of optical probes specific to tumor associated antigens such as the HER-2/neu receptor in aggressive breast cancers. Quantum dots and other fluorescent dyes coupled to peptides or other probes will greatly enhance our ability to detect cancers earlier and without ionizing radiation.     

Bone Marrow Stromal Antigen 2 (BST-2) DNA Is Demethylated in Breast Tumors and Breast Cancer Cells

Background

Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells.

Materials and Methods

We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines.

Results

We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA.

Conclusion

Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer.     

Selection of DNA aptamers for breast cancer

A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.     

乳腺肿块X线检查常见漏、误诊原因的病例分析

目的:分析乳腺X线检查的误漏诊原因,提高诊断准确性。方法:选择2011年3月至2013年12月来我院就诊的135例经乳腺X线摄影和病理检查证实的乳腺肿瘤患者为研究对象,将X线摄影结果与病理检查结果对比,进行回顾性的分析。结果:病理诊断72例良性肿瘤而X线误诊为恶性7例(误诊率9.72%);63例恶性肿瘤而X线误诊为良性5例(漏诊率7.93%)。结论:乳腺X线误诊与乳腺致密程度、患者年龄以及肿瘤形态相关。掌握拍片技术减少技术性误差,提高影像质量,诊断时仔细阅片并熟知各类型乳腺疾病的特征性X影像表象,并与临床相结合,增强责任心,可减少乳腺X线检查的误漏诊。     

Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer

BACKGROUND: We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. MATERIALS AND METHODS: Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. RESULTS: A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. CONCLUSIONS: Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.