Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

<p>Abstract</p> <p>Background</p> <p>Genetically engineered mouse models of mammary gland cancer enable the <it>in vivo </it>study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue.</p> <p>Methods</p> <p>We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging.</p> <p>Results</p> <p>In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice.</p> <p>Conclusion</p> <p>In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary.</p

Delivery of molecular-specific optical contrast agents for cancer biomarker detection in live cells and tissues

Molecular-specific optical contrast agents have shown promise as potential non-invasive probes for the detection of cancer and its precursors. The topical use of optical contrast agents in vivo has been hindered, however, by the difficulty of delivering macromolecules through mucosal tissue. My goal was to develop a robust strategy for the delivery of molecular-specific optical contrast agents into live cells and tissues. Specifically, I sought to: (1) develop an efficient and reproducible strategy for intracellular delivery of optical contrast agents into live cells, (2) evaluate the feasibility of targeting human telomerase reverse transcriptase (hTERT) in live cells and fresh tissues, and (3) translate intracellular delivery strategies for the topical permeation of multi-layer mucosal tissue. This dissertation describes the development of a surfactant-based strategy to effectively and reproducibly label cancer biomarkers in live cells and tissues. Triton-X100 was evaluated for its ability to deliver targeted and untargeted optical contrast agents to different cell compartments. My findings indicate that Triton-X100, when used at the appropriate concentration, can permeabilize a variety of live cells in a reproducible and reversible manner. To assess the usefulness of Triton-X100 for the delivery molecular-specific contrast agents, antibodies specific to hTERT were delivered into live permeabilized cells. The sensitivity of this approach was validated using cell lines that differentially express hTERT and paired clinically normal and abnormal human biopsies. The feasibility of enhancing tissue permeation with Triton-X100 was assessed in freshly excised mucosal specimens. The depth and rate of tissue permeation following topical Triton-X100 treatment was evaluated as function of optical probe size. Delivery of molecular-specific optical contrast agents was tested in xenograft tumor specimens co-treated with Triton-X100. These experiments revealed that Triton-X100 can facilitate simultaneous labeling of clinically relevant intracellular and extracellular biomarkers in a controlled, uniform manner. Together, these findings provide evidence that cell- and tissue-impermeant contrast agents can be delivered into mucosal tissue in a sufficiently controlled and uniform manner to allow for cancer biomarker detection. Further studies are proposed to establish the safety of Triton-X100 for topical use in vivo

Noninvasive Imaging Methods to Improve the Diagnosis of Oral Carcinoma and Its Precursors: State of the Art and Proposal of a Three-Step Diagnostic Process

Abstract: Oral squamous cell carcinoma (OSCC) is the most prevalent form of cancer of lips and oral cavity, and its diagnostic delay, caused by misdiagnosis at the early stages, is responsible for high mortality ratios. Biopsy and histopathological assessment are the gold standards for OSCC diagnosis, but they are time-consuming, invasive, and do not always enable the patient’s compliance, mainly in cases of follow-up with the need for more biopsies. The use of adjunctive noninvasive imaging techniques improves the diagnostic approach, making it faster and better accepted by patients. The present review aims to focus on the most consolidated diagnostic techniques, such as vital staining and tissue autofluorescence, and to report the potential role of some of the most promising innovative techniques, such as narrow-band imaging, high-frequency ultrasounds, optical coherence tomography, and in vivo confocal microscopy. According to their contribution to OSCC diagnosis, an ideal three-step diagnostic procedure is proposed, to make the diagnostic path faster, better, and more accurate

VIBRATIONAL SPECTROSCOPY FOR THE ASSESSMENT OF VULVAL DISEASE

Vibrational spectroscopic diagnostic techniques have significant potential to improve the care of women with benign, premalignant and malignant vulval diseases by reducing the reliance on traditional biopsy and histopathology. These techniques also have the potential to augment clinicians’ ability to differentiate different types of vulval disease at the time of surgery for neoplastic vulval disease. In addition, vibrational spectroscopic techniques offer the opportunity to assess molecular changes associated with the development of vulval cancer that are not apparent on routine histopathological assessment. The work outlined in this thesis evaluates the role of emerging techniques in vibrational spectroscopy to address this need within three key themes: 1. Developmentofavibrationalspectroscopicdiagnostictechniquetoreducethe reliance on traditional biopsy and histopathological diagnosis. 2. Developmentofavibrationalspectroscopicdiagnostictechniqueforimproving the delineation of disease margins at the time of surgery for pre-malignant and malignant vulval conditions. 3. Evaluation of a vibrational spectroscopic tool for augmenting and automating aspects of vulval histopathology. Raman spectroscopic mapping of 91 fresh frozen vulval tissue sections combined with multivariate spectral analysis was used to demonstrate that malignant vulval disease could be differentiated from non-neoplastic and premalignant vulval disease with a sensitivity of 97% and specificity of 78%. The technique was then tested in experimental conditions closer to in-vivo application, measuring spectra from 91 whole fresh frozen tissue blocks using microscope and probe Raman systems. This demonstrated the technique could differentiate malignant from non-neoplastic and premalignant vulval disease with sensitivities of 84% to 92% and specificities of 84% to 64% respectively. In a separate investigation vulval tissue blocks from 27 women with suspected lichen sclerosus underwent Raman spectroscopic point measurements. Multivariate analysis demonstrated Raman spectroscopy could be used to differentiate lichen sclerosus from other vulval disorders with a similar clinical appearance with a sensitivity of sensitivity of 91% and specificity of 80%. Fourier transform infrared (FTIR) spectroscopic mapping of 93 fixed paraffin embedded tissue sections was used to demonstrate that malignant vulval disease could be differentiated from non-neoplastic and premalignant with vulval disease with an approximate sensitivity of 100% and specificity of 79%. In addition FTIR spectroscopy was used to differentiate molecular changes in vulval intraepithelial neoplasia (VIN) and lichen sclerosus (LS) found in association with vulval squamous cell carcinoma (SCC). Analysis of FTIR spectroscopic tissue maps from 48 patients demonstrated the technique could differentiate LS associated with SCC with a sensitivity of approximately 100% and specificity of 84% and VIN associated with SCC with a sensitivity of approximately 100% and specificity 58%. This thesis demonstrates the considerable potential of vibrational spectroscopy in this clinical setting. The research has made significant progress in each of the three themes outlined above and indicates that further work is warranted to develop the techniques towards routine clinical application

Near real time confocal microscopy of Ex Vivo cervical tissue: detection of dysplasia

textRecent studies have shown the ability of confocal microscopy to noninvasively image cells in vivo in real time. This ability to visualize nuclei in vivo shows the potential of confocal microscopy to dramatically improve the prevention, detection and therapy of epithelial cancers. More exciting is the potential to quantitatively measure nuclear morphometry providing a basis to automate the cancer detection process. This dissertation describes studies exploring this potential in ex vivo cervical tissue using acetic acid as a nuclear contrast agent. First the use of acetic acid was demonstrated to improved contrast in confocal images of cervical tissue sufficiently to allow segmentation. Segmentation is robust throughout the epithelium in most normal tissue and upper portions of tissue diagnosed with severe dysplasia. Based upon this segmentation, quantitative feature measurements were extracted from confocal images of cervical tissue in a pilot study to determine if the features would aide in the detection of dysplasia. Simultaneously, a qualitative review of confocal images was performed by untrained reviewers and compared with clinical colposcopic impressions, the standard clinical tool aiding in dysplasia detection. The sensitivity and specificity of both the qualitative (95% and 69%) and quantitative (100% and 91%) review were improved compared to colposcopic review (91% and 62%). Finally the ability of confocal microscopy to produce 3D images was explored as a further means to improve dysplasia detection. Based upon Beer’s equation for light attenuation, the scattering coefficient was extracted from 3D image sets of ex vivo cervical tissue and compared with histology from the same precancerous lesion. The results suggested a possible correlation between high scattering values and the presence of dysplasia. Quantitative 3D features were also extracted from 3D image sets and correlated with the presence of CIN 2/3. Increased separation between normal and CIN 2/3 biopsies was produced using the 3D features as compared to the 2D. More importantly, when additional information (scattering coefficient) is combined with the 2D features, the ability to distinguish between normal and CIN 2/3 is 100% accurate in this small sample set.Electrical and Computer Engineerin

Curcumin Nanoformulation for Cervical Cancer Treatment

Cervical cancer is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation and poor bioavailability, its translational and clinical use has been limited. To improve these clinically relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation (Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR effectively inhibits cell growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis. Moreover, Nano-CUR effectively reduced the tumor burden in a pre-clinical orthotopic mouse model of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an effective therapeutic modality for cervical cancer