Growth Pattern Analysis of Murine Lung Neoplasms by Advanced Semi-Automated Quantification of Micro-CT Images

Computed tomography (CT) is a non-invasive imaging modality used to monitor human lung cancers. Typically, tumor volumes are calculated using manual or semi-automated methods that require substantial user input, and an exponential growth model is used to predict tumor growth. However, these measurement methodologies are time-consuming and can lack consistency. In addition, the availability of datasets with sequential images of the same tumor that are needed to characterize in vivo growth patterns for human lung cancers is limited due to treatment interventions and radiation exposure associated with multiple scans. In this paper, we performed micro-CT imaging of mouse lung cancers induced by overexpression of ribonucleotide reductase, a key enzyme in nucleotide biosynthesis, and developed an advanced semi-automated algorithm for efficient and accurate tumor volume measurement. Tumor volumes determined by the algorithm were first validated by comparison with results from manual methods for volume determination as well as direct physical measurements. A longitudinal study was then performed to investigate in vivo murine lung tumor growth patterns. Individual mice were imaged at least three times, with at least three weeks between scans. The tumors analyzed exhibited an exponential growth pattern, with an average doubling time of 57.08 days. The accuracy of the algorithm in the longitudinal study was also confirmed by comparing its output with manual measurements. These results suggest an exponential growth model for lung neoplasms and establish a new advanced semi-automated algorithm to measure lung tumor volume in mice that can aid efforts to improve lung cancer diagnosis and the evaluation of therapeutic responses

Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

<p>Abstract</p> <p>Background</p> <p>Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.</p> <p>Methods</p> <p>Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.</p> <p>Results</p> <p>We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.</p> <p>Conclusions</p> <p>Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.</p

Lung Volume Calculation in Preclinical MicroCT: A Fast Geometrical Approach

Lung; Preclinical imaging; VolumePulmón; Imágenes preclínicas; VolumenPulmó; Imatges preclíniques; VolumIn this study, we present a time-efficient protocol for thoracic volume calculation as a proxy for total lung volume. We hypothesize that lung volume can be calculated indirectly from this thoracic volume. We compared the measured thoracic volume with manually segmented and automatically thresholded lung volumes, with manual segmentation as the gold standard. A linear regression formula was obtained and used for calculating the theoretical lung volume. This volume was compared with the gold standard volumes. In healthy animals, thoracic volume was 887.45 mm3, manually delineated lung volume 554.33 mm3 and thresholded aerated lung volume 495.38 mm3 on average. Theoretical lung volume was 554.30 mm3. Finally, the protocol was applied to three animal models of lung pathology (lung metastasis and transgenic primary lung tumor and fungal infection). In confirmed pathologic animals, thoracic volumes were: 893.20 mm3, 860.12 and 1027.28 mm3. Manually delineated volumes were 640.58, 503.91 and 882.42 mm3, respectively. Thresholded lung volumes were 315.92 mm3, 408.72 and 236 mm3, respectively. Theoretical lung volume resulted in 635.28, 524.30 and 863.10.42 mm3. No significant differences were observed between volumes. This confirmed the potential use of this protocol for lung volume calculation in pathologic models

Preclinical evaluation of nanoparticle enhanced breast cancer diagnosis and radiation therapy

Triple negative breast cancer (TNBC) is an aggressive type of cancer which makes up 15-20% of all newly diagnosed cases, lacking the main target molecules for tumor specific treatment. Surgery or systemic therapy by chemotherapy are frequently used in the clinic and combined with radiation therapy to improve locoregional control in breast cancer patients after surgery. With a poor prognosis, there is a clear need to explore new treatment options for TNBC. The aim of the here presented PhD project was to evaluate the feasibility to enhance the biological effect of radiation therapy and increase tumor contrast for diagnosis by applying an in vivo microCT imaging system in combination with barium nanoparticles (BaNPs) in a pH8N8 WAP-T-NP8 mouse model for TNBC. Characterization of the BaNPs revealed strong x-ray attenuation and no toxic effects in different cancer and normal cell lines. Furthermore, irradiation of cancer cells using low energy x-rays in the keV range by a microCT resulted in a significant reduction on colony formation capability. In vitro, this low energy irradiation effect on clonogenic tumor cell survival was enhanced in the presence of BaNPs. Next, a subcutaneous lung cancer mouse model in immunodeficient mice and an orthotopic syngeneic mouse model for breast cancer was applied for further in vivo evaluation. Once the treatment plan was optimized regarding the applied x-ray doses and the frequency of irradiation, low energy radiation therapy within a classical in vivo microCT significantly reduced tumor growth or even resulted in shrinkage of the tumors without visible side effects and weight loss in comparison to untreated controls. However, the intratumoral application of BaNPs was not able to increase the irradiation effect on tumor growth kinetics. This might be in part due to inhomogeneous distribution of BaNPs within the tumor observed by microCT imaging. K-edge subtraction imaging as well as x-ray fluorescence of explanted tumor samples confirmed these findings. To localize the BaNPs in 3D to specific sites within the tumor environment and to detect morphological alterations within the tumor due to irradiation in proximity to BaNPs an ex-vivo imaging based analytic platform was established, utilizing co-registration of microCT and histology data. This imaging approach co-localized BaNPs with CD68 positive phagocytic cells and revealed a non-uniform distribution of the BaNPs within the tumor, however with no signs of locally enhanced radiation effects. Furthermore, antibody functionalized BaNPs were generated for systemic application. Analysis of biodistribution revealed that EpCAM labeled BaNPs did not reach the tumor after intra-venous administration, but accumulated in liver and spleen, demonstrated by a strong CT contrast within these organs. In summary, I showed that low energy radiation therapy by applying an in vivo microCT significantly reduced tumor volumes in comparison to untreated tumors in a syngeneic breast cancer tumor mouse model resembling TNBC. However, BaNPs while enhancing the effectiveness of irradiation on tumor cells in vitro, did not improve the irradiation effect on tumor growth in vivo.2021-07-1

A fully automated micro‑CT deep learning approach for precision preclinical investigation of lung fibrosis progression and response to therapy

: Micro-computed tomography (µCT)-based imaging plays a key role in monitoring disease progression and response to candidate drugs in various animal models of human disease, but manual image processing is still highly time-consuming and prone to operator bias. Focusing on an established mouse model of bleomycin (BLM)-induced lung fibrosis we document, here, the ability of a fully automated deep-learning (DL)-based model to improve and speed-up lung segmentation and the precise measurement of morphological and functional biomarkers in both the whole lung and in individual lobes. µCT-DL whose results were overall highly consistent with those of more conventional, especially histological, analyses, allowed to cut down by approximately 45-fold the time required to analyze the entire dataset and to longitudinally follow fibrosis evolution and response to the human-use-approved drug Nintedanib, using both inspiratory and expiratory μCT. Particularly significant advantages of this µCT-DL approach, are: (i) its reduced experimental variability, due to the fact that each animal acts as its own control and the measured, operator bias-free biomarkers can be quantitatively compared across experiments; (ii) its ability to monitor longitudinally the spatial distribution of fibrotic lesions, thus eliminating potential confounding effects associated with the more severe fibrosis observed in the apical region of the left lung and the compensatory effects taking place in the right lung; (iii) the animal sparing afforded by its non-invasive nature and high reliability; and (iv) the fact that it can be integrated into different drug discovery pipelines with a substantial increase in both the speed and robustness of the evaluation of new candidate drugs. The µCT-DL approach thus lends itself as a powerful new tool for the precision preclinical monitoring of BLM-induced lung fibrosis and other disease models as well. Its ease of operation and use of standard imaging instrumentation make it easily transferable to other laboratories and to other experimental settings, including clinical diagnostic applications

EPITHELIAL TO MESENCHYMAL TRANSITION AS A PREDICTOR OF RESPONSE TO POLO-LIKE KINASE 1 INHIBITION-INDUCED APOPTOSIS IN NON-SMALL CELL LUNG CARCINOMA

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Outcomes are poor for patients with recurrent, advanced or metastatic NSCLC. Polo-like kinase 1 (PLK1), involved in the regulation of mitotic processes and the response to DNA damage, is overexpressed in NSCLC. Inhibiting PLK1 may be an effective treatment for NSCLC patients as it is involved in the mechanisms of resistance to several chemotherapy drugs. PLK1 inhibition or knock-down has various effects in cancer cells, including mitotic arrest, apoptosis, and senescence. Predictive biomarkers have not been identified to select those patients who are likely to respond to PLK1 inhibitors although a small subset of NSCLC patients respond well to single agent therapy. Our lab found that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than the epithelial cell lines in vitro. The induction of an epithelial phenotype using miR-200 expression increased resistance to PLK1 inhibition, whereas the induction of a mesenchymal phenotype using ZEB1 expression or TGF-β increased PLK1 inhibition–induced apoptosis. To elucidate the mechanisms of resistance to PLK1 inhibition, our lab compared gene and protein expression in sensitive and resistant NSCLC cell lines and we identified β-Catenin, SMAD4 and PDK1 to be differentially regulated between epithelial and mesenchymal NSCLC cell lines after PLK1 inhibition. We tested the role of β-Catenin, SMAD4 and PDK1 in PLK1 inhibition induced apoptosis in NSCLC. Here, we demonstrate that mesenchymal NSCLC tumors are more sensitive to PLK1 inhibition compared to epithelial NSCLC in vivo in patient derived-xenograft (PDX) models as well as orthotopic mouse models in which the EMT properties are manipulable by modulating the miR200/ZEB1 axis. To facilitate analysis of these in vivo studies, we developed a novel semi-automated method of metastatic lung tumor burden calculation from computed tomography images by the calculation of the mass of the thoracic cavity. This method takes into account the aggregate tumor metastases in the thoracic cavity which significantly accounts for tumor burden in lung adenocarcinoma and provides details about the dynamic processes that occur in vivo over time

Radiation-induced lung damage promotes breast cancer lung-metastasis through CXCR4 signaling

Radiotherapy is a mainstay in the postoperative treatment of breast cancer as it reduces the risks of local recurrence and mortality after both conservative surgery and mastectomy. Despite recent efforts to decrease irradiation volumes through accelerated partial irradiation techniques, late cardiac and pulmonary toxicity still occurs after breast irradiation. The importance of this pulmonary injury towards lung metastasis is unclear. Preirradiation of lung epithelial cells induces DNA damage, p53 activation and a secretome enriched in the chemokines SDF-1/CXCL12 and MIF. Irradiated lung epithelial cells stimulate adhesion, spreading, growth, and (transendothelial) migration of human MDA-MB-231 and murine 4T1 breast cancer cells. These metastasis-associated cellular activities were largely mimicked by recombinant CXCL12 and MIF. Moreover, an allosteric inhibitor of the CXCR4 receptor prevented the metastasis-associated cellular activities stimulated by the secretome of irradiated lung epithelial cells. Furthermore, partial (10%) irradiation of the right lung significantly stimulated breast cancer lung-specific metastasis in the syngeneic, orthotopic 4T1 breast cancer model. Our results warrant further investigation of the potential pro-metastatic effects of radiation and indicate the need to develop efficient drugs that will be successful in combination with radiotherapy to prevent therapy-induced spread of cancer cells

Development of a Carbon Nanotube-Based Micro-CT and its Applications in Preclinical Research

Due to the dependence of researchers on mouse models for the study of human disease, diagnostic tools available in the clinic must be modified for use on these much smaller subjects. In addition to high spatial resolution, cardiac and lung imaging of mice presents extreme temporal challenges, and physiological gating methods must be developed in order to image these organs without motion blur. Commercially available micro-CT imaging devices are equipped with conventional thermionic x-ray sources and have a limited temporal response and are not ideal for in vivo small animal studies. Recent development of a field-emission x-ray source with carbon nanotube (CNT) cathode in our lab presented the opportunity to create a micro-CT device well-suited for in vivo lung and cardiac imaging of murine models for human disease. The goal of this thesis work was to present such a device, to develop and refine protocols which allow high resolution in vivo imaging of free-breathing mice, and to demonstrate the use of this new imaging tool for the study many different disease models. In Chapter 1, I provide background information about x-rays, CT imaging, and small animal micro-CT. In Chapter 2, CNT-based x-ray sources are explained, and details of a micro-focus x-ray tube specialized for micro-CT imaging are presented. In Chapter 3, the first and second generation CNT micro-CT devices are characterized, and successful respiratory- and cardiac-gated live animal imaging on normal, wild-type mice is achieved. In Chapter 4, respiratory-gated imaging of mouse disease models is demonstrated, limitations to the method are discussed, and a new contactless respiration sensor is presented which addresses many of these limitations. In Chapter 5, cardiac-gated imaging of disease models is demonstrated, including studies of aortic calcification, left ventricular hypertrophy, and myocardial infarction. In Chapter 6, several methods for image and system improvement are explored, and radiation therapy-related micro-CT imaging is present. Finally, in Chapter 7 I discuss future directions for this research and for the CNT micro-CT.Doctor of Philosoph