Name: ultrasonographic evaluation of breast cancer-related lymphedema
Uploaded: 2017-01-12T13:00:00
Description: Watch this Scientific Journal Video about Ultrasonographic Evaluation of Breast Cancer-related Lymphedema at JoVE.com

The authors have nothing to acknowledge.

All of the procedures were reviewed and approved by the Institutional Review Board of Chungnam National University Hospital.
1. Measurement with Ultrasonography
<ol>
	<li>Set the posture of the patient.
	<ol>
		<li>Lay the patient&#39;s arm in a supine position, with both arms on the bed.</li>
	</ol>
	</li>
	<li>Determine the measurement sites on the patient&#39;s upper limb.
	<ol>
		<li>Mark in the mid-point of the wrist crease, the mid-point between the medial and lateral epicondyles at the level of the elbow,...

<ol>
	<li>Lasinski, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+decongestive+therapy+for+treatment+of+lymphedema.">Complete decongestive therapy for treatment of lymphedema.</a> Seminars in Oncology Nursing. 29 (1), 20-27 (2013).</li><li>Clark, B., Sitzia, J., Harlow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+risk+of+arm+oedema+following+treatment+for+breast+cancer:+a+three-year+follow-up+study.">Incidence and risk of arm oedema following treatment for breast cancer: a three-year follow-up study.</a> Q J Med. 98 (5), 343-348 (2005).</li><li>Hayes, S. C., Speck, R. M., Reimet, E., Stark, A., Schmitz, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+effect+of+weight+lifting+on+lymphedema+following+breast+cancer+differ+by+diagnostic+method:+results+from+a+randomized+controlled+trial.">Does the effect of weight lifting on lymphedema following breast cancer differ by diagnostic method: results from a randomized controlled trial.</a> Breast Cancer Res Treat. 130 (1), 227-234 (2011).</li><li>Fu, M. R., Ridner, S. H., Armer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-breast+cancer+lymphedema:+part+1.">Post-breast cancer lymphedema: part 1.</a> Am J Nurs. 109 (7), 48-54 (2009).</li><li>Rockson, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+in+the+Evaluation+of+Breast+Cancer-Related+Lymphedema.">Ultrasonography in the Evaluation of Breast Cancer-Related Lymphedema.</a> Lymphatic Research and Biology. 14 (1), 1 (2016).</li><li>Suehiro, K., 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=Significance+of+Ultrasound+Examination+of+Skin+and+Subcutaneous+Tissue+in+Secondary+Lower+Extremity+Lymphedema.">Significance of Ultrasound Examination of Skin and Subcutaneous Tissue in Secondary Lower Extremity Lymphedema.</a> Ann Vasc Dis. 6 (2), 180-188 (2013).</li><li>Lee, J. H., 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=Ultrasonographic+Evaluation+of+Therapeutic+Effects+of+Complex+Decongestive+Therapy+in+Breast+Cancer-Related+Lymphedema.">Ultrasonographic Evaluation of Therapeutic Effects of Complex Decongestive Therapy in Breast Cancer-Related Lymphedema.</a> Ann Rehabil Med. 37 (5), 683-689 (2013).</li><li>DiSipio, T., Rye, S., Newman, B., Hayes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+unilateral+arm+lymphoedema+after+breast.">Incidence of unilateral arm lymphoedema after breast.</a> Lancet Oncol. 14 (6), 500-515 (2013).</li><li>Stanton, A. W., 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=Lymphatic+drainage+in+the+muscle+and+subcutis+of+the+arm+after+breast+cancer+treatment.">Lymphatic drainage in the muscle and subcutis of the arm after breast cancer treatment.</a> Breast Cancer Res Treat. 117 (3), 549-557 (2008).</li><li>Godoy, M. F. G., Pereira, M. R., Oliani, A. H., Godoy, J. M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergic+Effect+of+Compression+Therapy+and+Controlled+Active+Exercises+Using+a+Facilitating+Device+in+the+Treatment+of+Arm+Lymphedema.">Synergic Effect of Compression Therapy and Controlled Active Exercises Using a Facilitating Device in the Treatment of Arm Lymphedema.</a> Int J Med Sci. 9 (4), 280-284 (2012).</li><li>Cormie, P., Pumpa, K., Galv&#227;o, D. A., Turner, E., Spry, N., Saunders, C., Zissiadis, Y., Newton, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+safe+and+efficacious+for+women+with+lymphedema+secondary+to+breast+cancer+to+lift+heavy+weights+during+exercise:+a+randomised+controlled+trial.">Is it safe and efficacious for women with lymphedema secondary to breast cancer to lift heavy weights during exercise: a randomised controlled trial.</a> J Cancer Surviv. 7 (3), 413-424 (2013).</li><li>Bok, S. K., Jeon, Y., Hwang, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonographic+evaluation+of+the+effects+of+progressive+resistive+exercise+in+breast+cancer-related+lymphedema.">Ultrasonographic evaluation of the effects of progressive resistive exercise in breast cancer-related lymphedema.</a> Lymphatic Research and Biology. 14 (1), 18-24 (2016).</li><li>Milne, H. M., Wallman, K. E., Gordon, S., Courneya, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+combined+aerobic+and+resistance+exercise+program+in+breast+cancer+survivors:+a+randomized+controlled+trial.">Effects of a combined aerobic and resistance exercise program in breast cancer survivors: a randomized controlled trial.</a> Breast Cancer Res Treat. 108 (2), 279-288 (2008).</li><li>Schmitz, K. H., 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=Weight+lifting+for+women+at+risk+for+breast-cancer-related+lymphedema.">Weight lifting for women at risk for breast-cancer-related lymphedema.</a> JAMA. 304 (24), 2699-2705 (2010).</li><li>Kiel, K. D., Rademacker, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-stage+breast+cancer:+Arm+edema+after+wide+excision+and+breast+irradiation.">Early-stage breast cancer: Arm edema after wide excision and breast irradiation.</a> Radiology. (1), 279-283 (1996).</li></ol>

Evaluations of BCRL can be conducted through various techniques, including arm circumferences tape measurement, water displacement measurement, bioimpedance spectroscopy, perometry (optoelectronic volumeter), lymphoscintigraphy, clinician diagnosis, or swelling self-reports by the patient. However, there are no standards for the measurement of BCRL; the diagnosis and duration of treatment can be determined empirically in a number of ways, depending upon the clinic.
Clinically, measuring arm ci...

Lymphedema is defined as a condition of localized protein-rich fluid retention and tissue swelling caused by a compromised lymphatic system1,2. As lymphedema progresses, fibrocytes and/or adipocytes proliferate in the affected areas, leading to changes in the texture of the skin and subcutaneous tissue and an increased vulnerability to bacterial and fungal infections3,4. The observation of these changes in various parts of the extremities may further elucidate the severity and extent of lymphedema. The subfascial lymphatic system contributes to the severity of lymphedema more than the epifascial lymphatic system.
Recent research has mainly focused on the use of ultrasonography in the diagnosis of lymphedema5,6. Ultrasonography is a relatively inexpensive method to observe soft-tissue characteristics. A previous study reported that lymphedema can be reliably diagnosed with ultrasonography by comparing the thickness of the skin and subcutaneous tissue and also by evaluating the compliance of the subcutaneous tissue in a clinical setting7,8. In addition, ultrasonography is widely used in the clinic because of its easy accessibility, safety from radiation exposure, and inexpensiveness5,6. In spite of these benefits, studies on ultrasonography for lymphedema have only been focused on diagnosis, not on evaluation of therapeutic intervention.
Therefore, the current study aimed to determine not only the usefulness of ultrasonography in elucidating the effects of PRE&#160;on BCRL, but also whether ultrasonography has a clinical significance as a follow-up test in confirming treatment efficacy. This study was conducted to establish the protocol for ultrasounds performed to examine lymphedema.

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		<tr>
			<td>ACUSON S2000</td>
			<td>Siemens</td>
			<td>10041461</td>
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		</tr>
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			<td>Eco Gel 99</td>
			<td>Seung Won Medical Corp.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrasonographic evaluation of breast cancer-related lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study included 32 patients diagnosed with BCRL. The thickness of muscle and subcutaneous tissue and the circumferences of proximal and distal upper limbs were measured at baseline, 4 weeks, and 8 weeks after PRE. Examiners measured the circumference of upper limbs with a tape measure, and then the thickness of the muscle and subcutaneous tissue were measured by ultrasonography. Patients were randomly divided into a PRE group and a non-PRE group. Participants were reminded to not reve...

Watch this Scientific Journal Video about Ultrasonographic Evaluation of Breast Cancer-related Lymphedema at JoVE.com

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

The overall goal of this procedure is to diagnose breast cancer-related lymphedema and evaluate the therapeutic effect of resistive exercise by using ultrasonography to measure the thickness of subcutaneous tissue. Begin by laying the patient's arm in a supine position with both arms on the bed. Use a pen to mark the midpoint of the wrist crease, the midpoint of the medial and lateral epicondyles at the level of the elbow, in the bicipital groove.Then connect these points using a ruler. Next, mark the first target area 10 centimeters proximal to the elbow point along the line between the elbow and bicipital groove. In the second target area, 10 centimeters distal to the elbow point along the line between the elbow and the wrist.Then, switch on the ultrasound system. Enter the 2-D mode of the soft tissue via the keyboard. Set the depth to five centimeters and choose the 14L5 linear array transducer.Apply sufficient gel to the ultrasound transducer and place it perpendicularly to the upper limb ventral axis. The short axis view to capture the image where the thickness of the gel is at least one centimeter and the soft tissue contours are not distorted. Next, select the calibrator and the software via the keyboard of the ultrasounds system to draw a line between the muscle and the subcutaneous tissue.Then, measure the thickness of the muscle, defined at the distance between the highest point on the boundary of the bone to the highest point on the boundary portion of the fascia. Finally, measure the thickness of the subcutaneous tissue, defined as the distance from the skin to the fascia. After measuring the upper limb muscle and subcutaneous tissue thickness at baseline, give the patient a 5 kilogram dumbbell and have him or her put on a compression stocking.Ask the patient to perform the progressive resistant exercises, or PRE, in the following order. Dumbbell fly, tricep extension, one arm bent-over row, bicep curls, dumbbell side raise, and lifting the arms forward. Next, instruct the patient to complete the six exercises twice a day according to the following schedule.Five times each during the first week, 10 times each during the second week, 15 times each during the third week, 20 times each during the forth week, and 25 times each during the fifth through eighth weeks. Inform the subject to return at four and eight weeks later to measure the thickness of the subcutaneous tissue and muscle of the upper limb again. Finally, after all measurements are collected, analyze the data by comparing the difference between both arms using a paired T test.Compare the changes in subcutaneous tissue and muscle thickness using a repeated measures ANOVA. According to the tape measurement, arm circumference did not significantly change in the PRE group after four weeks of exercise. However, both proximal and distal arm circumferences showed a significant reduction after eight weeks.As measured by ultrasonography in the PRE group, the thickness of muscle in the distal part showed a significant increase at four weeks and eight weeks. and the proximal part demonstrated a significant increase in thickness at eight weeks. Lastly, in the PRE group, the thickness of subcutaneous tissue in the affected upper limb significantly decreased after eight weeks.No significant difference in these parameters was observed in the control group.

ultrasonographic-evaluation-of-breast-cancer-related-lymphedema

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on self-report questionnaires which are subject to report and recall biases. Objective measurements using a handheld dynamometer, or handgrip device, have been shown in recent studies to correlate significantly with subjective self-reported fatigue scores. However, variations of both the handgrip fatigue test and fatigue index calculations exist in the literature. The lack of standardized methods limits the utilization of the handgrip fatigue test in the clinical and research settings. In this study, we provide detailed methods for administering the physical fatigue test and calculating the fatigue index. These methods should supplement existing self-reported fatigue questionnaires and help clinicians assess fatigue symptom severity in an objective and quantitative manner.

Simple and accessible methods were developed to measure the motor aspect of cancer-related fatigue objectively and quantitatively. We describe, in detail, ways to administer the physical fatigue test using a simple handgrip device as well as methods to calculate fatigue indices.