This research was supported in part by the Intramural Research Program of the National Cancer Institute.

1. Cell culture maintenance of dormant and metastatic tumor cell lines
<ol>
 <li>Grow dormant (D2OR/ MCF7/K7M2-AS.46) and metastatic tumor cells (D2A1/ MDA-MB-231/ K7M2) in 10 cm culture plates containing Dulbecco's Modified Eagle's Medium (DMEM) high glucose and 10% Fetal bovine serum (FBS) and antibiotics. Once the cells reach 70-80% confluence, proceed to the following assays.</li>
</ol>
2. Cell proliferation assay of dormant (quiescent) and metastatic (proliferating) tumor cells cultured in a 3D-BME system
Culturing dormant/metastatic cells in the 3D system
<ol>
 <li>Thaw Cultrex growth factor-reduced Basement Membrane Extract (BME) in 4&deg;C refrigerator a night before carrying out the assay. Note the BME should be handled on ice at all times.</li>
 <li>On the following day, place a 96 well plate on a tray of ice inside a laminar hood. Coat each well with 50-100&mu;l of ice cold BME using a dispenser with a syringe. Make sure no bubbles are formed in the wells. Place the 96 well plate coated with BME in a humidified incubator with 5% CO2 at 37&deg;C for 30 minutes.</li>
 <li>In the meantime aspirate the media from the dormant and or metastatic tumor cells (prepared in section 1). Rinse culture plates with 10 ml phosphate buffered saline, ph 7.4 (PBS). Aspirate the PBS and add 2ml trypsin pre-warmed at 37&deg;C, to the culture plates. Incubate the plates in a humidified 5% CO2 at 37&deg;C, for 5 min.</li>
 <li>Transfer cells to a 15 ml conical tube containing 5ml of DMEM high glucose supplemented with 10% FCS and antibiotics and count the cells.</li>
 <li>Spin down the total cell number to be cultured in a tissue culture centrifuge at a speed of 1500g, at room temperature for 5 min. In our assays we prepare 2X103 cells /well for each cell line or time point to be examined. However, this may vary depending upon the cell lines used.</li>
 <li>Carefully aspirate the supernatant. Note, in most cases the pellet is not visible. Therefore, leave some media behind. Tap the bottom of the 15ml conical tube with your fingers to ensure that a suspension of single cells is obtained. Re-suspend the pellet with DMEM low glucose with antibiotics supplemented with 2% FCS + 2% BME (assay media). 100 &mu;l of the assay media should be added for every 2x103 cells. Triturate the cells many times with a 5ml pipette to ensure that a single cell suspension is maintained.</li>
 <li>Plate a 100&mu;l of the cell mixture per well on top of the 96 well BME coated plate. For background evaluation (in section 2.8) plate in addition 100&mu;l per well of only the assay media on top of the 96 well BME coated plate. Incubate the cultured 96 well plates in a humidified 5% CO2 incubator at 37&deg;C. Cells should be re-fed every 4 days with the assay media.</li>
</ol>
Proliferation assay:
<ol start="8">
 <li>Proliferation assay of the cells: add to the wells at the desired time points 20 &mu;l of the Cell Titer 96 AQueous One Solution Cell Proliferation assay kit. Incubate in a humidified 5% CO2 incubator at 37&deg;C for 2h. Using an Elisa Plate Reader record the absorbance at 490nm. For background evaluation and subtraction, add 20&mu;l of the Cell Titer 96 AQueous One Solution Cell Proliferation assay kit to wells pre coated with BME and only overlaid with assay media. Using an Elisa Plate Reader record the absorbance at 490 nm.</li>
</ol>
3. Immunofluorescent staining for cell signaling molecules in dormant (quiescent) tumor cells and/or metastatic (proliferating) tumor cells
Culturing dormant/metastatic cells in the 3D system for immunfluorescence staining
*The following protocol is a modification of a 3D culture protocol published by Debnath J et al 18.
<ol>
 <li>Prepare BME as described in section 2.1. The following day: place an 8-chamber glass slide system on a tray of ice inside a laminar hood. Coat each well with 50ul of ice-cold BME using a 200&mu;l pipetman. Make sure BME is spread evenly and no bubbles are formed in the wells. Place the 8 chamber glass slide coated with BME in a humidified 5% CO2 at 37&deg;C for 20 min.</li>
 <li>Harvest dormant and or metastatic cells from section 1 and prepare to culture as described in section 2.3- 2.4. Collect the total number of cells to be cultured in a 15 ml conical tube. We prepare 5 x103 cells / well for each cell line and time point to be examined. Spin down the cells in a tissue culture centrifuge at a speed of 1500g, at room temperature for 5 min. Aspirate the supernate carefully. Note that the pellet is not visible; therefore leave some media behind. Tap the bottom of the 15ml conical tube with your fingers to ensure that a single cell suspension is obtained. Re-suspend the pellet with assay media. 400&mu;l of the assay media should be added for every 5x103 cells. Triturate the cells many times with 5ml pipette. This step is very important to ensure that the single cell suspension is maintained.</li>
 <li>Plate 400&mu;l of the cell mixture per well on top of each of the 8 chambers coated with BME. Incubate the cultured 8 chambers glass slide system in a humidified 5% CO2 incubator at 37&deg;C. Cells should be re-fed every 4 days with the assay media.</li>
</ol>
Immunofluorescence staining:
<ol start="4">
 <li>At the desired time points, aspirate the upper layer of the media and add 200&mu;l of the fixative containing 4% Paraformaldehyde (PFA), 5% sucrose and 0.1% Triton X-100 and incubate at room temperature for 5 minutes. Aspirate the fixative and add 200&mu;l of 4% PFA containing 5% sucrose and incubate at room temperature for 25 minutes.</li>
 <li>Aspirate the fixative; add 400 &mu;l of phosphate buffered saline (PBS) to each well. Incubate for 10 minutes at room temperature. Aspirate the PBS and add 400 &mu; PBS containing 0.05% Tween 20 for 10 minutes at room temperature.</li>
 <li>Block the fixed cells at room temperature with 200&mu;l of either 10% donkey serum or with 3% BSA for 1hour (blocking solution to be used should be determined empirically for each primary antibody).</li>
 <li>Aspirate the blocking solution and add 200&mu;l of the primary antibody (dilution should be determined empirically for each primary antibody to be used). Dilute the primary antibody in 10% donkey serum if 10% donkey serum was used for blocking or dilute primary antibody in 3% BSA if 3% BSA blocking solution was used. Incubate with the primary antibody overnight at 4&deg;C.</li>
 <li>Aspirate the antibody, wash the wells with 400&mu;l PBS for 15 minutes and repeat twice. Aspirate the PBS and add 200&mu;l of donkey anti-respective- IgG conjugated to rhodamine red (dilution should be determined empirically), cover the 8 chamber slide with aluminum foil and incubate for 1 hour at room temperature.</li>
 <li>Wash the wells with 400&mu;l PBS (3x15 minutes each wash). Aspirate PBS. Mounted with VECTASHIELD mounting medium with DAPI. Dry slides for 40 minutes at room temperature in the dark. Slides can be kept for 1 week at 4&deg;C. Store the slides in the dark. Image slides by Confocal microscopy.</li>
</ol>
4. Representative Results:
An example of a proliferation analysis of the dormant D2.0R and metastatic D2A1 tumor cells in the 3D culture is shown in Figure 1A. D2.0R cells are dormant (quiescent) through the entire experimental 14 day culture period whereas the highly metastatic D2A1 cells remain dormant only for four to six days after which they begin to proliferate. During the initial dormant phase, many cells remain solitary in the 3-D culture (Figure 1B; day 4) whereas other non-proliferating cells form multi-cellular spheroids. The transition of D2A1 cells from a dormant to proliferative state in 3-D culture (Figure 1B; day 12) is associated with dramatic changes in cell morphology. Hence, this assay can be used to test what factor/s may trigger dormant D2.0R cells to emerge from their dormant state and what factor/s may prevent D2A1 cells to transition from their dormant state. Figure 2 is an example of an agent preventing D2A1 cells to transition from dormant to a proliferative state. As illustrated in Figure 2, treatments of D2A1 cells with a specific inhibitor of myosin light chain kinase (ML- 7) maintained D2A1 cells in a dormant state.
Cell signaling in the dormant and proliferating tumor cells cultured in the 3D system can be studied by immunofluorescence staining for cell signaling molecules. As illustrated in Figure 3 a significant increase in myosin light chain phosphorylation in D2A1 cells (red staining) followed by reorganization of the f-actin filaments forming actin stress fibers (green staining) occurs during their transition from dormancy (1-4 days) to proliferation (day 7). However, blocking myosin light chain kinase activity in D2A1 cells by shRNA or specific drug (ML-7) retains D2A1 cells in a dormant state and results in inhibition of myosin light chain phosphorylation and f-actin stress fiber organization (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/2914/2914fig1.jpg" /> 
Figure 1. in vitro model to study solitary tumor cell dormancy and the switch to metastatic growth. A) Proliferation of dormant D2.0R and metastatic D2A1 in 3-D Cultrex BME, n=8 (mean &plusmn; SE). Representative results of three experiments (* p&le;0.05). B) Light microscopy images of D2.0R and D2A1 cells cultured in 3-D Cultrex BME magnification x20. Figure modified from Barkan et al 17.
<img alt="Figure 2" src="/files/ftp_upload/2914/2914fig2.jpg" /> 
Figure 2. Preventing the switch of D2A1 cells from dormancy (quiescence) to proliferation in the 3D culture system by inhibition of myosin light chain kinase (MLCK). Time course of D2A1 cell proliferation cultured in 3-D Cultrex BME , n=8 (mean &plusmn;SE). Cells were untreated (control), or treated with a specific inhibitor of MLCK (ML-7; 5 &mu;M) for 48 hr beginning on culture day 5. Figure modified from Barkan et al 17.
<img alt="Figure 3" src="/files/ftp_upload/2914/2914fig3.jpg" /> 
Figure 3. Myosin light chain phosphorylation followed by f-actin reorganization during the switch of D2A1 cells from dormancy to proliferative growth. D2A1 cells were cultured in 3-D Cultrex BME on 8 chamber glass slide. Cells were fixed and stained with DAPI (blue) for nuclear localization, phalloidin (green) for f-actin and with an antibody against the phosphorylated form of myosin light chain (MLC-p) (red), as indicated at various time points. Merge of f-actin, and MLC-p staining (yellow). Expression of MLC-p was increased during the transition of D2A1 cells from dormancy (days1-4) to proliferative growth (day 7) followed by actin stress fiber formation (arrows). Confocal microscopy, magnification x63. White bar equals 20 microns. Figure modified from Barkan et al 17.
<img alt="Figure 4" src="/files/ftp_upload/2914/2914fig4.jpg" /> 
Figure 4. Inhibition of myosin light chain kinase (MLCK) mediated f-actin stress fiber formation in D2A1 cells. D2A1 Cells were untreated (control), or treated with inhibitor for MLCK (ML-7; 5 &mu;M), for 48 hr beginning on culture day 5, or treated with scrambled or MLCK shRNA and stained for the phosphorylated form of myosin light chain (MLC-p) (red), f- actin (green), and nuclei (blue). Merge of f-actin, and MLC-p staining (yellow). Confocal microscopy, magnification x63. White bar equals 20 microns.

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No conflicts of interest declared.

The underlying mechanisms that maintain disseminated tumor cells in a dormant state or result in their transition to metastatic growth remain largely unknown. This phenomenon has been extremely difficult to study in human patients 4,12 and few preclinical models have been developed to address this issue. Nevertheless, some in vivo and ex-vivo model systems for tumor dormancy have been characterized (reviewed in 1,12). However, the in vivo models for tumor dormancy can p...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Invitrogen </td>
			<td>11885-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10091-148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced 3-D Cultrex Basement Membrane Extract</td>
			<td>Trevigen Inc.</td>
			<td>&nbsp;</td>
			<td>Protein concentration between 14-15mg/ml</td>
		</tr>
		<tr>
			<td>D2.0R and D2A1 cell lines</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5,19</td>
		</tr>
		<tr>
			<td>K7M2 and K7M2AS1.46 cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>20</td>
		</tr>
		<tr>
			<td>MCF-7 and MDA-MB-231 breast cancer cells</td>
			<td>ATCC</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>An 8 chamber glass slide system</td>
			<td>(Lab -TEK, Thermo scientific)</td>
			<td>177402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Titer 96 AQueous One Solution cell proliferation assay kit</td>
			<td>Promega</td>
			<td>G3580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VECTASHIELD mounting medium with DAPI</td>
			<td>Vector Laboratories Inc.</td>
			<td>H-1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elisa Plate Reader</td>
			<td>Bio-Tec</td>
			<td>&nbsp;</td>
			<td>Record 490nm</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss-LSM-510</td>
			<td>&nbsp;</td>
			<td>Magnification x63</td>
		</tr>
	</tbody>
</table>

an in vitro system to study tumor dormancy and the switch to metastatic growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

Watch this Scientific Journal Video about An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth at JoVE.com

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

an-vitro-system-to-study-tumor-dormancy-switch-to-metastatic

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15.8K Views.  University of Haifa. Un modificato 3-D in vitro del sistema è presentato in cui caratteristiche di crescita di diverse linee cellulari tumorali in membrana basale ricostituita in correlazione con il comportamento inattivo o proliferativa delle cellule tumorali in un sito secondario metastatico In vivo.

Video: An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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 ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...