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
<ol>
	<li>Include patients if (1) they have pathologically confirmed thyroid cancer and require surgical treatment; (2) they have extensive local tumor infiltration, such as T3-T4 (American Joint Committee on Cancer TNM staging, eighth edition), or if metastatic lesions have invaded important structures such as the trachea, esophagus, and large vessels; (3) they and their family members volunteer for computer-aided 3D visualization; and (4) they had no contraindications to anesthesia.</li>
	<li>Exclude patients if they do not undergo surgical treatment.</li>
</ol>
2. Imaging acquisition
<ol>
	<li>Obtain plain and enhanced CT (including venous and arterial phases) images of the patients using a 256-layer spiral CT system. The scanning parameters are as follows: 120 kV, 120 mA, 512 x 512 matrix, 0.625 mm layer thickness, 150 HU threshold, and 10-20-s arterial scan delay.</li>
	<li>Obtain the scan data from the CT system in DICOM format.</li>
</ol>
3. Computer-aided 3D modeling
<ol>
	<li>Import the data into the 3D visualization software (Figure 1A).
	<ol>
		<li>Click on the Open button to select the document containing the patient data in DICOM format. Import the data into the software.</li>
		<li>Process the data for Gaussian smoothing if the original data contain a lot of image noise (Figure 1B). Select the data with the right mouse button, and then click on the Gaussian Smoothing button.</li>
	</ol>
	</li>
	<li>Reconstruct different structures in the target area (chest and neck) individually.
	<ol>
		<li>Select Different Models (for example, skin and bone) in the software according to the structure to be reconstructed (Figure 2A).</li>
		<li>Set the Color, Maximum Threshold, and Minimum Threshold based on the reconstructed structure on CT (Figure 2B). Set different thresholds for the bone and skin. Adjust the upper and lower thresholds based on the observed preview effect (Figure 2C).</li>
		<li>Click on the Calculation button to complete the preliminary 3D model reconstruction (Figure 2D).</li>
	</ol>
	</li>
	<li>Modify the segmented data.
	<ol>
		<li>Once the segmentation data of structures such as the blood vessels, skin, and bones are obtained (Figures 3A-C), use the Smoothing Algorithm button to optimize the segmented data and ensure the reconstructed saw-tooth edges match the real tissue.</li>
		<li>Then, use the One-Click Navigation button to locate the 2D and 3D images (Figure 3D), and determine whether the segmentation effect was accurate. Use the Pen or Brush tool to fix the incorrect layers (Figure 3E). 
		&#8203;NOTE: The 3D modeling is achieved after obtaining the segmentation data of all the structures.</li>
	</ol>
	</li>
</ol>
4. Preoperative evaluation
<ol>
	<li>View the 3D model, and pay close attention to the tumor volume and location and the relationships between the tumor and the adjacent tissues using the Magnification, Rotation, Tissue Transparency, and Separation functions and a combination of various structures. For example, observe the extent of tumor invasion in the common carotid artery, esophagus, and trachea.</li>
	<li>Determine the scope of surgical resection, the degree of functional impairment after resection, and the postoperative adjuvant therapy plan based on the 3D model evaluation. Implement effective and intuitive doctor-patient communication to satisfy the patient&#39;s expectations and explain the surgeon&#39;s treatment plan.</li>
</ol>
5. Surgery
<ol>
	<li>Remove the tumor according to the preoperative plan and the intraoperative observations of the tumor and the affected vital organs.</li>
	<li>Perform tumor-reducing surgery with intraoperative labeling for postoperative adjuvant therapy in the absence of a repair plan.</li>
	<li>Repair defects caused by the resection, and perform functional reconstruction as necessary based on the operative plan and intraoperative situation.</li>
</ol>

<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. 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The authors have nothing to disclose.

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 ...

computer-aided-three-dimensional-visualization-treatment-locally

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

459 Views.  Affiliated Cancer Hospital of University of Electronic Science and Technology of China. 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.

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

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 ...

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 ...

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex procedures. The high-quality 3-D visual, multijoint arm and natural tremor filtration allow the surgeon to expose and dissect more accurately with minimal invasion. Total mesorectal excision is the standard surgical technique for the treatment of resectable rectal cancer. To reduce the lateral recurrence rate, lateral pelvic lymph node dissection can be performed, as it is a safe and feasible procedure for locally advanced middle-low rectal cancer with a high possibility of metastasis to the lateral lymph nodes. However, the complexity of the anatomic structures and the high postoperative complication rate limit its application. Recently, several surgeons have increasingly used robotic techniques for total mesorectal excision and lateral pelvic lymph node dissection. Compared with open and laparoscopic surgery, the robotic technique has several advantages, such as less blood loss, fewer blood transfusions, minimal trauma, shorter postoperative hospitalization, and quicker recovery. A robotic approach is generally regarded as a reasonable alternative for complicated procedures such as lateral pelvic lymph node dissection, although there are a limited number of high-quality prospective randomized controlled studies reporting direct evidence. Here, we provide the detailed steps of robot-assisted total mesorectal excision and lateral pelvic lymph node dissection performed at the First Affiliated Hospital of Xi&#39;an Jiaotong University.

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex ...

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

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer