Name: computer-aided three-dimensional visualization in the treatment of locally advanced thyroid cancer
Uploaded: 2023-06-09T14:20:00
Description: Watch this Scientific Journal Video about Computer-Aided Three-Dimensional Visualization in the Treatment of Locally Advanced Thyroid Cancer at JoVE.com

The authors have no acknowledgments.

This study protocol was approved by the Ethics Committee of Sichuan Cancer Hospital (Approval date: September 27, 2019). All the procedures involving human participants were performed in accordance with the ethical standards of the institutional and national research committees, as well as the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from all the patients before surgery.
1. Inclusion and exclusion criteria
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	<li>Include...

<ol>
	<li>Zheng, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+in+China,+2016.">Cancer incidence and mortality in China, 2016.</a> Journal of the National Cancer Center. 2 (1), 1-9 (2022).</li><li>Haugen, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+American+thyroid+association+management+guidelines+for+adult+patients+with+thyroid+nodules+and+differentiated+thyroid+cancer:+The+American+thyroid+association+guidelines+task+force+on+thyroid+nodules+and+differentiated+thyroid+cancer.">2015 American thyroid association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: The American thyroid association guidelines task force on thyroid nodules and differentiated thyroid cancer.</a> Thyroid. 26 (1), 1 (2016).</li><li>Haddad, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+carcinoma,+Version+2.2022,+NCCN+Clinical+practice+guidelines+in+oncology.">Thyroid carcinoma, Version 2.2022, NCCN Clinical practice guidelines in oncology.</a> Journal of the National Comprehensive Cancer Network: JNCCN. 20 (8), 925-951 (2022).</li><li>Wang, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operative+management+of+locally+advanced,+differentiated+thyroid+cancer.">Operative management of locally advanced, differentiated thyroid cancer.</a> Surgery. 160 (3), 738-746 (2016).</li><li>Shindo, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+invasive+well-differentiated+thyroid+cancer:+an+American+Head+and+Neck+Society+Consensus+Statement.+AHNS+Consensus+Statement.">Management of invasive well-differentiated thyroid cancer: an American Head and Neck Society Consensus Statement. AHNS Consensus Statement.</a> Head &amp; Neck. 36 (10), 1379-1390 (2014).</li><li>Li, X., Song, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+strategies+in+surgical+management+of+differentiated+thyroid+cancer+invading+upper+areodigestive+tracts.">Principles and strategies in surgical management of differentiated thyroid cancer invading upper areodigestive tracts.</a> Chinese Journal of Otorhinolaryngology. Head and Neck Surgery. 52 (06), 478-480 (2017).</li><li>Yamanaka, J., Saito, S., Fujimoto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+preoperative+planning+using+virtual+segmental+volumetry+on+liver+resection+for+hepatocellular+carcinoma.">Impact of preoperative planning using virtual segmental volumetry on liver resection for hepatocellular carcinoma.</a> World Journal of Surgery. 31 (6), 1251-1257 (2007).</li><li>Fang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+three+dimensional+visualization+technique+assisted+hepatectomy+for+the+treatment+of+primary+liver+cancer.">Efficacy of three dimensional visualization technique assisted hepatectomy for the treatment of primary liver cancer.</a> Chinese Journal of Surgery. 53 (8), 574-579 (2015).</li><li>Gong, Y., Mao, X., Yang, B., Jiang, Q., Yin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+progress+of+digital+medicine+in+orthopedic+surgery.">Application progress of digital medicine in orthopedic surgery.</a> Chinese Journal of Joint Surgery (Electronic Edition). 2, 266-270 (2018).</li><li>Study Group of Pancreatic Surgery in Chinese Society of Surgery of Chinese Medical Association, Pancreatic Committee of Chinese Research Hospital Association, Digital Medicine Branch of Chinese Medical Association, Digital Medicine Committee of Chinese Research Hospital Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+of+precise+diagnosis+and+treatment+for+pancreatic+head+cancer+using+three-dimensional+visualization+technology.">Expert consensus of precise diagnosis and treatment for pancreatic head cancer using three-dimensional visualization technology.</a> Chinese Journal of Surgery. 55 (12), 881-886 (2017).</li><li>Lyshchik, A., Drozd, V., Reiners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three-dimensional+ultrasound+for+thyroid+volume+measurement+in+children+and+adolescents.">Accuracy of three-dimensional ultrasound for thyroid volume measurement in children and adolescents.</a> Thyroid. 14 (2), 113-120 (2004).</li><li>Yi, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two-+and+three-dimensional+sonography+for+the+prediction+of+the+extrathyroidal+extension+of+papillary+thyroid+carcinomas.">Comparison of two- and three-dimensional sonography for the prediction of the extrathyroidal extension of papillary thyroid carcinomas.</a> Korean Journal of Internal Medicine. 31 (2), 313-322 (2016).</li><li>Han, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+and+combined+application+of+two-dimensional+and+three-dimensional+real-time+shear+wave+elastography+in+diagnosis+of+thyroid+nodules.">Comparisons and combined application of two-dimensional and three-dimensional real-time shear wave elastography in diagnosis of thyroid nodules.</a> Journal of Cancer. 10 (9), 1975-1984 (2019).</li><li>Cai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+thyroid+three-dimensional+reconstruction+for+endoscopic+thyroidectomy.">Application of thyroid three-dimensional reconstruction for endoscopic thyroidectomy.</a> Journal of Laparoscopic Surgery. 17 (6), 408-411 (2012).</li><li>Chen, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+computer-aided+design+(CAD)+and+three-dimensional+(3D)+visualization+technologies+in+the+diagnosis+and+treatment+of+refractory+thyroid+tumors.">Application of computer-aided design (CAD) and three-dimensional (3D) visualization technologies in the diagnosis and treatment of refractory thyroid tumors.</a> Cancer Management and Research. 12, 6887-6894 (2020).</li><li>Zanon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+virtual+planning+for+nodule+resection+in+solid+organs:+A+systematic+review+and+meta-analysis.">Three-dimensional virtual planning for nodule resection in solid organs: A systematic review and meta-analysis.</a> Surgical Oncology. 38, 101598 (2021).</li><li>Chinese Society of Clinical Oncology (CSCO) diagnosis and treatment guidelines for persistent/recurrent and metastatic differentiated thyroid cancer working group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+Society+of+Clinical+Oncology+(CSCO)+diagnosis+and+treatment+guidelines+for+persistent/recurrent+and+metastatic+differentiated+thyroid+cancer+2018+(English+version).">Chinese Society of Clinical Oncology (CSCO) diagnosis and treatment guidelines for persistent/recurrent and metastatic differentiated thyroid cancer 2018 (English version).</a> Chinese Journal of Cancer Research. 31 (1), 99-116 (2019).</li><li>Chinese Thyroid Association, Chinese College of Surgeons, Chinese Medical Doctor Association, the Society of Thyroid Cancer of China Anti-Cancer Association, Chinese Research Hospital Association Thyroid Disease Committee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+on+the+diagnosis+and+treatment+of+medullary+thyroid+carcinoma+(2020+edition).">Expert consensus on the diagnosis and treatment of medullary thyroid carcinoma (2020 edition).</a> Chinese Journal of Practical Surgery. 40 (9), 2012 (2020).</li><li>Chinese Society of Digital Medicine, Liver Cancer Committee of Chinese Medical Doctor Association, Clinical Precision Medicine Committee of Chinese Medical Doctor Association, Digital Intelligent Surgery Committee of Chinese Research Hospital Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice+guidelines+for+precision+diagnosis+and+treatment+of+complex+liver+tumor+guided+by+three-dimensional+visualization+technology+(version+2019).">Clinical practice guidelines for precision diagnosis and treatment of complex liver tumor guided by three-dimensional visualization technology (version 2019).</a> Journal of South Medical University. 40 (3), 297-307 (2020).</li><li>Scharpf, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+management+of+recurrent+thyroid+cancer:+An+American+Head+and+Neck+Society+consensus+statement:+AHNS+consensus+statement.">Comprehensive management of recurrent thyroid cancer: An American Head and Neck Society consensus statement: AHNS consensus statement.</a> Head &amp; Neck. 38 (12), 1862-1869 (2016).</li></ol>

For recurrent and metastatic differentiated thyroid carcinoma (DTC), surgical treatment is still preferred17. The 5 year disease-specific survival rate of patients with DTC and R0 resection is 94.4%, which is significantly higher than that of patients with R1 resection (67.9 %)2. Achieving disease control in the neck is crucial for attaining a better quality of life and disease-specific survival for patients4. Medullary thyroid carcinoma is mainly tr...

Thyroid cancer is the seventh most common malignancy in China1, and surgery is the most important treatment method2,3. Complete resection of the tumor is strongly associated with high survival rates and a good quality of life in patients with locally advanced thyroid cancer3,4; however, this type of resection is challenging. The neck contains important organs and tissues, such as the trachea, esophagus, and common carotid artery. Resection for advanced thyroid cancer is even more risky and difficult considering the proximity of such tumors to important organs and large blood vessels in the neck and mediastinum5,6. Thus, adequate preoperative evaluation is necessary.
Currently, computed tomography (CT), magnetic resonance (MRI), and color Doppler ultrasonography, which are widely used in clinical settings, provide a two-dimensional (2D) view, which limits the evaluation of the tumor volume, boundaries, and relationships with important surrounding structures7,8. Substantial clinical experience and efficient trial and error are required before surgeons can translate 2D images into 3D space. Computer-aided 3D visualization can use 2D imaging to create a more intuitive 3D model that can be used for preoperative planning and treatment plan selection, thereby making doctor-patient communication more intuitive and reducing doctor-patient disagreements. Although the model provides 3D visualization, it is intangible. This 3D-guided preoperative evaluation and preparation can shorten the surgical time and reduce the surgical risks. The 3D approach has been widely used in hepatobiliary surgery, orthopedics, and oral and maxillofacial surgery9,10. In thyroid cancer, 3D visualization is currently used to assist in ultrasonic diagnosis and in the formulation of surgical plans11,12,13,14,15.
Therefore, we believe that 3D visualization can be conveniently applied to the diagnosis and treatment of locally advanced thyroid cancer. This visualization method includes CT acquisition, computer-aided 3D modeling, and preoperative evaluation using 3D models. The 3D models can be used to determine surgical difficulties, surgical risks, and the potential postoperative functional status. Surgeons can engage in detailed doctor-patient communication, surgical plan formulation, and the corresponding surgical preparation16. Furthermore, this method can provide an adequate preoperative assessment of patients, reduce the surgical risks, and improve patient satisfaction without increasing patient trauma.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brilliance 256-layer spiral CT system</td>
			<td>Philips Healthcare, Andover, MA, USA</td>
			<td>N/A</td>
			<td>Used for plain and enhanced CT imaging</td>
		</tr>
		<tr>
			<td>3D-Matic digital medical software application</td>
			<td>Anhui King Star Digital S&#38;T Co. Ltd.</td>
			<td>N/A</td>
			<td>Used for computer-aided 3D visualization reconstruction</td>
		</tr>
	</tbody>
</table>

computer-aided three-dimensional visualization in the treatment of locally advanced thyroid cancer

The diagnosis and treatment of locally advanced thyroid carcinoma are challenging. The challenge lies in the evaluation of the tumor scope and the formulation of an individualized treatment plan. Three-dimensional (3D) visualization has a wide range of applications in the field of medicine, although there are limited applications in thyroid cancer. We previously applied 3D visualization for the diagnosis and treatment of thyroid cancer. Through data collection, 3D modeling, and preoperative evaluation, we can obtain 3D information regarding the tumor outline, determine the extent of tumor invasion, and conduct adequate preoperative preparation and surgical risk assessment. This study aimed to demonstrate the feasibility of 3D visualization in locally advanced thyroid cancer. Computer-aided 3D visualization can be an effective method for accurate preoperative evaluation, the development of surgical methods, shortening the surgical time, and reducing the surgical risks. Furthermore, it can contribute to medical education and doctor-patient communication. We believe that the application of 3D visualization technology can improve outcomes and quality of life in patients with locally advanced thyroid cancer.

From December 2017 to July 2021, 23 patients with locally advanced thyroid cancer underwent 3D modeling. Of these 23 patients, 4 were excluded from surgery owing to surgical risks, and the remaining 19 patients were treated with surgery following 3D modeling (Table 1). All 19 patients had locally advanced thyroid cancer, including 14 for whom this was the initial diagnosis, 16 who had varying degrees of dyspnea, and 18 who had large tumors in the neck (primary thyroid tumor or metastatic lymph node) that...

Watch this Scientific Journal Video about Computer-Aided Three-Dimensional Visualization in the Treatment of Locally Advanced Thyroid Cancer at JoVE.com

Computer-Aided Three-Dimensional Visualization in the Treatment of Locally Advanced Thyroid Cancer

In diagnosing and treating locally advanced thyroid cancer, the application of computer-aided three-dimensional reconstruction can provide additional information regarding the tumor scope and anatomic characteristics, thereby assisting in risk assessment and surgical planning.

The diagnosis and treatment of locally advanced thyroid carcinoma are challenging. The challenge lies in the evaluation of the tumor scope and the ...

The computer aided three dimensional reconstruction can provide information regarding the tumor scope and anatomy characteristics, thereby assisting in risk assessment and surgical plan. The computer aided three-dimensional reconstruction can provide information for diagnosing and treating local advanced thyroid cancer. To begin, obtain plain and enhanced CT images of the patients using a 256 layer spiral CT system.Obtain the scan data from the CT system in DICOM format. Then import the data into the 3D visualization software by clicking on the Open button. If the data contains image noise, process the data for Gaussian smoothing by first selecting the data with the right mouse button, and then clicking on the Gaussian smoothing button.Next, select the tab Different Models in the software according to the structure to be reconstructed, such as blood vessels or the skin. Set the color, maximum threshold and minimum threshold. Adjust the upper and lower thresholds based on the observed preview effect.Repeat the procedure to obtain the segmentation data of other structures such as the bones. Click on the calculation button to complete the preliminary 3D model reconstruction. Then use the smoothing algorithm button to optimize the segmented data and ensure that the reconstructed sawtooth edges match the real tissue.Then use the one click navigation button to locate the 2D and 3D images and determine whether the segmentation effect was accurate. Use the pen or brush tool to fix the incorrect layers. For preoperative evaluation, view the 3D model and evaluate the tumor volume and location.Using the magnification, rotation, tissue transparency and separation functions, evaluate the relationships between the tumor and the adjacent tissues using a combination of various structures. In the case of a 50 year old patient with thyroid carcinoma, initial evaluation using CT angiography suggested that a large number of vessels were involved in the tumor. Using CT data, the surgical team completed computer-aided 3D modeling of the tumor.Evaluation of the 3D model revealed the relationships between the tumor and the blood vessels, and further operative procedures were determined. It's important to check whether the three dimensional images is consistent with the CT images for successful reconstruction.

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Research

JoVE Journal

Medicine

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...