Name: integrating augmented reality tools in breast cancer related lymphedema prognostication and diagnosis
Uploaded: 2020-02-06T12:00:00
Description: Watch this Scientific Journal Video about Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis at JoVE.com

This study was approved by the Institutional Review Boards from the Authors&#39; Institutions. This protocol applies to node-positive (i.e., N&#62;1 according to the TNM staging system)14 breast cancers showing LVI and/or ENE.
1. Real time arm measurements
<ol>
	<li>Install the free downloadable application (e.g., Captevia) on a tablet computer.</li>
	<li>Take a marker of the highest-contrast color compared to the skin of the patient (e.g., white, black).
	<ol>
		<li>...

<ol>
	<li>Michelotti, A., 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=Tackling+the+diversity+of+breast+cancer+related+lymphedema:+Perspectives+on+diagnosis,+risk+assessment,+and+clinical+management.">Tackling the diversity of breast cancer related lymphedema: Perspectives on diagnosis, risk assessment, and clinical management.</a> Breast. 44, 15-23 (2018).</li><li>Noguchi, M., Yokoi, M., Nakano, Y., Ohno, Y., Kosaka, T., Singh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axillary+reverse+mapping+in+breast+cancer.">Axillary reverse mapping in breast cancer.</a> Radioisotopes - Applications in Bio-Medical Science. , (2011).</li><li>Wilke, L. G., 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=Surgical+complications+associated+with+sentinel+lymph+node+biopsy:+results+from+a+prospective+international+cooperative+group+trial.">Surgical complications associated with sentinel lymph node biopsy: results from a prospective international cooperative group trial.</a> Annals of Surgical Oncology. 13 (4), 491-500 (2006).</li><li>Taghian, N. R., Miller, C. L., Jammallo, L. S., O'Toole, J., Skolny, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphedema+following+breast+cancer+treatment+and+impact+on+quality+of+life:+a+review.">Lymphedema following breast cancer treatment and impact on quality of life: a review.</a> Critical Reviews in Oncology/Hematology. 92 (3), 227-234 (2014).</li><li>Dean, L. T., 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=34;It+still+affects+our+economic+situation&amp;#34;:+long-term+economic+burden+of+breast+cancer+and+lymphedema.">34;It still affects our economic situation&amp;#34;: long-term economic burden of breast cancer and lymphedema.</a> Supportive Care in Cancer. , (2018).</li><li>Invernizzi, 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=Lymphovascular+invasion+and+extranodal+tumour+extension+are+risk+indicators+of+breast+cancer+related+lymphoedema:+an+observational+retrospective+study+with+long-term+follow-up.">Lymphovascular invasion and extranodal tumour extension are risk indicators of breast cancer related lymphoedema: an observational retrospective study with long-term follow-up.</a> BMC Cancer. 18 (1), 935 (2018).</li><li>Invernizzi, 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=Breast+Cancer+Systemic+Treatments+and+Upper+Limb+Lymphedema:+A+Risk-Assessment+Platform+Encompassing+Tumor-Specific+Pathological+Features+Reveals+the+Potential+Role+of+Trastuzumab.">Breast Cancer Systemic Treatments and Upper Limb Lymphedema: A Risk-Assessment Platform Encompassing Tumor-Specific Pathological Features Reveals the Potential Role of Trastuzumab.</a> Journal of Clinical Medicine. 8 (2), (2019).</li><li>Kilbreath, S. 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=Risk+factors+for+lymphoedema+in+women+with+breast+cancer:+A+large+prospective+cohort.">Risk factors for lymphoedema in women with breast cancer: A large prospective cohort.</a> Breast. 28, 29-36 (2016).</li><li>Sun, F., 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=The+need+for+preoperative+baseline+arm+measurement+to+accurately+quantify+breast+cancer-related+lymphedema.">The need for preoperative baseline arm measurement to accurately quantify breast cancer-related lymphedema.</a> Breast Cancer Research and Treatment. 157 (2), 229-240 (2016).</li><li>Deltombe, T., 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=Reliability+and+limits+of+agreement+of+circumferential,+water+displacement,+and+optoelectronic+volumetry+in+the+measurement+of+upper+limb+lymphedema.">Reliability and limits of agreement of circumferential, water displacement, and optoelectronic volumetry in the measurement of upper limb lymphedema.</a> Lymphology. 40 (1), 26-34 (2007).</li><li>Tewari, N., Gill, P. G., Bochner, M. A., Kollias, J. <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+volume+displacement+versus+circumferential+arm+measurements+for+lymphoedema:+implications+for+the+SNAC+trial.">Comparison of volume displacement versus circumferential arm measurements for lymphoedema: implications for the SNAC trial.</a> ANZ Journal of Surgery. 78 (10), 889-893 (2008).</li><li>Cau, 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=Comparative+study+between+circumferential+method+and+laser+scanner+3D+method+for+the+evaluation+of+arm+volume+in+healthy+subjects.">Comparative study between circumferential method and laser scanner 3D method for the evaluation of arm volume in healthy subjects.</a> Journal of Vascular Surgery: Venous and Lymphatic Disorders. 4 (1), 64-72 (2016).</li><li>Hameeteman, M., Verhulst, A. C., Vreeken, R. D., Maal, T. J., Ulrich, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+stereophotogrammetry+in+upper-extremity+lymphedema:+An+accurate+diagnostic+method.">3D stereophotogrammetry in upper-extremity lymphedema: An accurate diagnostic method.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 69 (2), 241-247 (2016).</li><li>Amin, M. 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=.">.</a> AJCC Cancer Staging Manual. , (2017).</li><li>Kamper, S. 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=Global+Perceived+Effect+scales+provided+reliable+assessments+of+health+transition+in+people+with+musculoskeletal+disorders,+but+ratings+are+strongly+influenced+by+current+status.">Global Perceived Effect scales provided reliable assessments of health transition in people with musculoskeletal disorders, but ratings are strongly influenced by current status.</a> Journal of Clinical Epidemiology. 63 (7), 760-766 (2010).</li></ol>

The use of an accurate and reliable tool for limb volume measurement is mandatory in breast cancer survivors, given that an early diagnosis of BCRL is related to improved outcomes. In addition, the identification of high-risk individuals should consider not only clinical and surgical data but also tumor-specific pathological features. This study demonstrates the reproducibility and reliability of a new portable 3DLS device in upper limb volume measurement both in healthy subjects and in a BCRL women with high-risk featur...

Breast cancer related lymphedema (BCRL) is fluid retention in the upper extremity occurring after axillary surgery and/or radiation therapy in approximately 20-80% of breast cancer patients with lymph node metastases (N&#62;1)1,2,3. This condition results in swelling of the limb with subsequent reduced functionality, increased risk of comorbidities, frustration, anxiety, and depression4,5. BCRL is currently considered a long-standing threat for breast cancer survivors given that it can arise up to 9 years after surgery6.
BCRL pathogenesis is a subject of lively debate among breast cancer specialists. Hence, several studies have shown that it could be related not only to axillary interventions but also to systemic treatments, such as taxanes-based chemotherapy and anti-HER2 therapy7,8. Furthermore, there are recent lines of evidence to suggest that tumor-specific pathological characteristics are involved in its pathogenesis1,6,7. For this reason, the invasion of lymphovascular spaces at the periphery of the tumor by neoplastic cells (lymphovascular invasion, LVI) and of the extension of the metastasis beyond the lymph node capsule (extra-nodal extension, ENE) have been proposed as a complementary analysis for BCRL risk stratification6,7.
Despite the novel insights of BCRL biology, the arm volume measurement remains a cornerstone phase in the diagnosis and follow-up of these patients9. For this task, however, there are not widely-adopted guidelines. The measurement of multiple circumferences across the arm has been traditionally used to estimate its total volume using the truncated cone formula10. Despite its reliability, however, this circumferential method (CM) lack sensitivity and reproducibility, particularly in the case of arm shape irregularities11. Lately, augmented reality methods, such as three-dimensional laser scanner (3DLS), have emerged as promising tools for the measurement of the upper limb volume12. This technology is cheap, user-friendly, reproducible, and extremely precise also in the presence of gibbousness and swelling12,13.
The aim of this study was to assess the reliability of 3DLS in comparison to the circumferential method for BCRL diagnosis and quantification. Here, a protocol is detailed for real-time measurements of the upper limb volume by means of 3DLS in breast cancer patients at increased risk for BCRL.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Structure sensor + Captevia V3.1</td>
			<td>Rodin4D, Rodin SAS</td>
			<td></td>
			<td>Three dimensional laser scanner</td>
		</tr>
	</tbody>
</table>

integrating augmented reality tools in breast cancer related lymphedema prognostication and diagnosis

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

This was a pilot single-blind, randomized controlled study involving adult individuals. Inclusion criteria were the following: 1) young adults aged &#62;18 and &#60;45 years; 2) normal weight (body mass index, BMI, &#62;18 and &#60;25 kg/m2); 3) absence of any kind of skin lesion at upper limb level; 4) absence of trauma and/or any kind of condition able to modify arm structure and volume. The exclusion criteria: 1) cardiovascular comorbidities; 2) vascular diseases involving t...

Watch this Scientific Journal Video about Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis at JoVE.com

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

The laser scan three-dimensional device allows a quick and reproducible measurement of the upper limb volume without requiring direct contact with the skin of patients. In addition, the technology is inexpensive, user friendly, reproducible, and extremely precise even in the presence of gibbousness and swelling. Demonstrating the procedure will be Dr.Lorenzo Lippi from our research group.Begin by installing a free downloadable 3D laser scanning application on a tablet computer. Next, use a marker of the highest contrast color compared to the skin of the patient to mark the skin of the upper limb to be analyzed at five centimeter landmarks starting from the wrist to the elbow and from the elbow to the proximal portion of the arm. Draw circumferences around the upper limb to be analyzed at the level of each landmark and position the patient in an upright position in a room with sufficient space to move around them.Raise the patient's upper limb anteriorly by 90 degrees and ask the patient to remain in this position without moving for the entire duration of the scan. Link a 3D laser scanner device to the tablet and open the 3D laser scanner application. Enter the credentials of the patient into the patient database and click connection and plus to enter a new patient.When all of the patient information has been entered, click the 3D icon and use two fingers to adjust the distance and size of the cube on the screen to select the area to be examined. Click scan and start the three-dimensional scan, framing the upper limb of the patient on all of the planes of the space, rotating around the limb from all angles multiple times to optimize the quality of the acquired image. When the acquisition is complete, press done.Then revise the form to assess any acquisition defects or missing parts as necessary. Next, select the acquisition and press the icon to enter an email and send the file. On the desktop computer, download the data sent by email and insert a USB drive with the software license for the 3D laser scanner software.After opening the data file, view the scan and click create new patient. Under the file menu, select the patient file and select the scan. Click extract.Select the extracted scan and click cleaning. Next, select the image and orient the image in the space using the skin landmarks to cut off any segments included in the scan on the various axes as necessary. Validate the cut figure and name the shape obtained from the scan.Click create a new shape to orient the shape axis on the three planes of the space. Click next and wait for the generation of the final form and click end. Select the processed form and click open.The software will provide the total volume of the processed form. Then click volume to calculate the volume of the different sections and move and select the upper and lower margin of the section from which to extract the volume. In this representative study, 30 healthy male and female adults and 30 breast cancer female patients who later developed breast cancer-related lymphedema were evaluated.Arm volume measurements were performed using both the circumferential and 3D laser scanner methods. In both groups, both methods demonstrated high levels of inter and intraoperator reproducibility with a mean R squared of 0.99 for both techniques. Accordingly, the mean value of the 3D laser scanner volume calculations also showed a strong correlation with those from the circumferential method in both groups as confirmed by the Bland-Altman plot showing a high level of agreement and consistency between the two different measurements.Furthermore, although differences were observed before and after complex decongestive treatment for both techniques in the breast cancer patient group, the 3D laser scanner method correlation was higher than that observed for the circumferential method. This procedure can be used in any physiological or pathological condition that require a volumetric evaluation of any part of the body. The use of this technique in a clinical setting can solve several issues related to the standardization of volumetric measurements in breast cancer-related lymphedema.

integrating-augmented-reality-tools-breast-cancer-related-lymphedema

Research

JoVE Journal

Medicine

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Combining Augmented Reality and 3D Printing to Display Patient Models on a Smartphone

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) printing (3DP) opens new possibilities in clinical applications. Although these technologies have grown exponentially in recent years, their adoption by physicians is still limited, since they require extensive knowledge of engineering and software development. Therefore, the purpose of this protocol is to describe a step-by-step methodology enabling inexperienced users to create a smartphone app, which combines AR and 3DP for the visualization of anatomical 3D models of patients with a 3D-printed reference marker. The protocol describes how to create 3D virtual models of a patient&rsquo;s anatomy derived from 3D medical images. It then explains how to perform positioning of the 3D models with respect to marker references. Also provided are instructions for how to 3D print the required tools and models. Finally, steps to deploy the app are provided. The protocol is based on free and multi-platform software and can be applied to any medical imaging modality or patient. An alternative approach is described to provide automatic registration between a 3D-printed model created from a patient&rsquo;s anatomy and the projected holograms. As an example, a clinical case of a patient suffering from distal leg sarcoma is provided to illustrate the methodology. It is expected that this protocol will accelerate the adoption of AR and 3DP technologies by medical professionals.

Presented here is a method to design an augmented reality smartphone application for the visualization of anatomical three-dimensional models of patients using a 3D-printed reference marker.

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) ...

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke survivors. The diagnosis and characterization of these symptoms in individual patients can be challenging and often requires skilled clinical staff. Virtual reality (VR) presents an opportunity to develop novel assessment tools for patients with USN.
We aimed to design and build a VR tool to detect and characterize subtle USN symptoms, and to test the tool on subjects treated with inhibitory repetitive transcranial magnetic stimulation (TMS) of cortical regions associated with USN.
We created three experimental conditions by applying TMS to two distinct regions of cortex associated with visuospatial processing- the superior temporal gyrus (STG) and the supramarginal gyrus (SMG) - and applied sham TMS as a control. We then placed subjects in a virtual reality environment in which they were asked to identify the flowers with lateral asymmetries of flowers distributed across bushes in both hemispaces, with dynamic difficulty adjustment based on each subject's performance. 
We found significant differences in average head yaw between subjects stimulated at the STG and those stimulated at the SMG and marginally significant effects in the average visual axis.
VR technology is becoming more accessible, affordable, and robust, presenting an exciting opportunity to create useful and novel game-like tools. In conjunction with TMS, these tools could be used to study specific, isolated, artificial neurological deficits in healthy subjects, informing the creation of VR-based diagnostic tools for patients with deficits due to acquired brain injury. This study is the first to our knowledge in which artificially generated USN symptoms have been evaluated with a VR task.

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke ...