Name: development of a human preclinical model of osteoclastogenesis from peripheral blood monocytes co-cultured with breast cancer cell lines
Uploaded: 2017-09-13T15:00:00
Description: Watch this Scientific Journal Video about Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines at JoVE.com

We would like to thank Yibin Kang for providing the SCP2 cell line and Cristiano Verna for editorial assistance.

Human osteoclasts were differentiated from PBMCs of healthy donors who gave written informed consent to take part in the study. The study protocol was approved by the local Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
1. Osteoclast Differentiation
NOTE: Collect peripheral blood or buffy coats from healthy human donors in EDTA. Do not use less than 20 mL of peripheral blood. Buffy coats are preferable because...

<ol>
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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=Tumor+microenvironment+complexity:+emerging+roles+in+cancer+therapy.">Tumor microenvironment complexity: emerging roles in cancer therapy.</a> Cancer Res. 72 (10), 2473-2480 (2012).</li><li>Roodman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+bone+metastasis.">Mechanisms of bone metastasis.</a> N Eng J Med. 350 (12), 1655-1664 (2004).</li><li>Patel, L. R., Camacho, D. F., Shiozawa, Y., Pienta, K. J., Taichman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cancer+cell+metastasis+to+the+bone:+a+multistep+process.">Mechanisms of cancer cell metastasis to the bone: a multistep process.</a> Future Oncol. 7 (11), 1285-1297 (2011).</li><li>Chen, Y. C., Sosnoski, D. M., Mastro, A. M. <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+metastasis+to+the+bone:+mechanisms+of+bone+loss.">Breast cancer metastasis to the bone: mechanisms of bone loss.</a> Breast Cancer Res. 12 (6), 215 (2010).</li><li>Guise, T. A. <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+bone+metastases:+it's+all+about+the+neighborhood.">Breast cancer bone metastases: it's all about the neighborhood.</a> Cell. 154 (5), 957-959 (2013).</li><li>Ell, 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=Tumor-induced+osteoclast+miRNA+changes+as+regulators+and+biomarkers+of+osteolytic+bonemetastasis.">Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bonemetastasis.</a> Cancer Cell. 24 (4), 542-556 (2013).</li><li>Lu, X., 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=VCAM-1+promotes+osteolytic+expansion+of+indolent+bone+micrometastasis+of+breast+cancer+by+engaging+&#945;4&#946;1-positive+osteoclast+progenitors.">VCAM-1 promotes osteolytic expansion of indolent bone micrometastasis of breast cancer by engaging &#945;4&#946;1-positive osteoclast progenitors.</a> Cancer Cell. 20 (6), 701-714 (2011).</li><li>Wang, 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=The+osteogenic+niche+promotes+early-stage+bone+colonization+of+disseminated+breast+cancer+cells.">The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.</a> Cancer Cell. 27 (2), 193-210 (2015).</li><li>Liverani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF-1+blockade+impairs+breast+cancer+osteoclastogenic+potential+in+co-culture+systems.">CSF-1 blockade impairs breast cancer osteoclastogenic potential in co-culture systems.</a> Bone. 66, 214-222 (2014).</li><li>Mercatali, 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=The+effect+of+everolimus+in+an+in+vitro+model+of+triple+negative+breast+cancer+and+osteoclasts.">The effect of everolimus in an in vitro model of triple negative breast cancer and osteoclasts.</a> Int J Mol Sci. 1 (11), e1827 (2016).</li><li>Glantschnig, H., Fisher, J. E., Wesolowski, G., Rodan, G. A., Reszka, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF,+TNFalpha+and+RANK+ligand+promote+osteoclast+survival+by+signaling+through+mTOR/S6+kinase.">M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.</a> Cell DeathDiffer. 10 (10), 1165-1177 (2003).</li><li>Sugatani, T., Hruska, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/Akt2+and+mammalian+target+of+rapamycin/Bim+play+critical+roles+in+osteoclast+differentiation+and+survival,+respectively,+whereas+Akt+is+dispensable+for+cell+survival+in+isolated+osteoclast+precursors.">Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors.</a> J Biol Chem. 280 (5), 3583-3589 (2005).</li><li>Bertoldo, 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=Targeting+bone+metastatic+cancer:+role+of+the+mTOR+pathway.">Targeting bone metastatic cancer: role of the mTOR pathway.</a> Biochim Biophys Acta. 1845 (2), 248-254 (2014).</li><li>Kang, Y., Siegel, P. M., Shu, W., Drobnjak, M., Kakonen, S. M., Cord&#243;n-Cardo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-549 (2003).</li><li>Simone, V., Ciavarella, S., Brunetti, O., Savonarola, A., Cives, M., Tucci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everolimus+restrains+the+paracrine+pro-osteoclast+activity+of+breast+cancer+cells.">Everolimus restrains the paracrine pro-osteoclast activity of breast cancer cells.</a> BMC Cancer. 14 (15), 692 (2015).</li></ol>

Preclinical in vitro models studying the mechanisms of crosstalk between cancer cells and bone cells are needed to identify mechanisms of bone metastasis that can be used to create new therapeutic strategies. We developed a fully human in vitro model of osteoclastogenesis from human peripheral blood (Figure 3). During optimization of the methodology, a number of critical points were identified and resolved. The first concerned the quantity of mononuclear cells to seed. The ...

Bone is a common site of metastasis for different types of primary tumors such as prostate, lung and breast cancer, with 20 - 25% of patients developing bone metastases during the course of disease1,2,3. In particular, 70% of breast cancer patients carry evidence of bone metastasis at death4. Tumor and stromal cell interaction is essential for cancer progression in both primary cancer and secondary lesions. In the bone microenvironment, osteolytic bone metastases from breast cancer depend on the establishment of a pathological vicious cycle occurring between cancer cells, bone cells, and the bone microenvironment. Cancer cells disrupt bone balance, increasing bone resorption5,6,7.
In normal and pathological conditions, osteoclasts are the cells responsible for bone resorption, whereas osteoblasts, in depositing new matrix, are responsible for new bone formation8. Osteoclast activity is regulated by osteoblasts through the expression of RANKL, which binds to its receptor RANK on the pre-osteoclast surface, inducing pre-osteoclast fusion, a necessary process for differentiation into mature osteoclasts. The induction of osteoclastogenesis increases bone resorption. A large number of in vivo studies have significantly improved our understanding of bone metastasis formation9,10,11. Breast cancer cells from the primary tumor and in the bone microenvironment perturb bone homeostasis, promoting osteoclastogenesis and bone resorption8. In this scenario, all the molecular interactions that occur between cancer cells and osteoclasts are of crucial importance. As already mentioned, the mechanism of bone metastasis formation has been elucidated in in vivo mice models. However, in addition to the need for the approval of all in vivo animal experiments by the Ethics Committee, there are several other drawbacks to performing in vivo experiments including high costs and time-consuming methods. Several authors have combined preclinical in vivo and in vitro models of osteoclastogenesis using a murine line of pre-osteoclasts called RAW246.79,10,11. The drawbacks of this model stem from the fact that the cells are already committed to becoming pre-osteoclasts and are not of human origin. For these reasons, translational research could greatly benefit from the availability of in vitro fully human preclinical models to study bone cancer cell interactions.
We optimized a method of osteoclastogenesis in vitro starting from human peripheral blood samples12,13. Osteoclasts derive from monocytes, which are present, albeit to a small degree, in peripheral blood samples. Mononuclear cells are first separated from the erythrocytes and granulocytes present in whole blood by Ficoll density gradient; they are then selected thanks to their ability to adhere to plastic substrate, unlike lymphocytes. After seeding, cells are cultured for 14 days. MCSF and RANKL are the GFs required by monocytes to differentiate first into macrophages and then into osteoclasts14,15. MCSF is needed for the entire duration of the assay, whereas RANKL is used to induce the differentiation process in the late stages of osteoclastogenesis. In the early phase of differentiation, MCSF helps monocytes proliferate and survive14,15. During the second part of osteoclastogenesis, cells fuse together and mature as osteoclasts, showing the characteristic distribution of Actin F in rings and expressing specific markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor (CTR)14,15. Our method consists of adding MCSF to the monocyte culture for the first 7 days of the experiment and a combination of MCSF and RANKL from days 7 to 14. At the end of the experiment, osteoclastogenesis is analyzed by counting the differentiated cells, as detailed below.
The monocyte cultures induced to differentiate by GFs form the basis of our preclinical model. We optimized a co-culture system without GFs to better understand the osteoclastogenic power of breast cancer cells. We first developed a model of indirect co-cultures by adding a medium (80% &#945; -Minimal Essential Medium (&#945;-MEM) and 20% conditioned medium collected from a culture of breast cancer cells that were about 90% confluent to cells undergoing differentiation12. The conditioned medium (not collected under serum deprivation conditions) was collected after 24 hours and mixed with fresh medium at a proportion of 1:4. The conditioned medium induced significant osteoclast differentiation with respect to the negative control. However, as the information on the reciprocal interaction between cancer cells and bone cells is lost when using indirect co-cultures, we improved our system by carrying out direct co-cultures. We seeded cancer cells in 0.4 &#181;M inserts and placed them in wells where mononuclear cells were plated. Using this method, cells share the same medium and exchange secreted proteins. We thus created a fully human preclinical model of osteoclastogenesis induced by cancer cells13.
This system is extremely versatile and can be used for different research purposes, e.g., in pharmacological studies investigating the role of drugs in bone metastasis. Our model makes it possible to study the efficacy and mechanisms of action of bone-targeted therapies and/or antitumor drugs in the bone microenvironment in the presence of cancer cells13. Designing the experiments with the correct controls, i.e., cancer cells and osteoclasts cultured individually, makes it easier to understand the impact of the co-culture on drug activity. This approach becomes even more interesting when the drug being studied targets both cancer cells and osteoclasts, e.g., everolimus16. This model can also be used to identify new pathways of interaction between cancer cells and bone cells.

<table>
	<tbody>
		<tr>
			<td>&#945;MEM</td>
			<td>Euroclone</td>
			<td>BE12-169F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life-technologies</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life-technologies</td>
			<td>ECS0180DPR</td>
			<td></td>
		</tr>
		<tr>
			<td>hMCSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>Storage indications must be respected</td>
		</tr>
		<tr>
			<td>hRANKL</td>
			<td>Peprotech</td>
			<td>310-01</td>
			<td>Storage indications must be respected</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma Aldrich</td>
			<td>387 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphocyte separation media</td>
			<td>Biowest</td>
			<td>L0560-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood cell lysing buffer</td>
			<td>SIgma</td>
			<td>11814389001 
			ROCHE</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>EuroClone</td>
			<td>COD. ECB3052D</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% aqueous solution, EM grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 cell line</td>
			<td>ATCC</td>
			<td>CRM-HTB-267</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF7</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3470-Clear</td>
			<td>These inserts are for 24-well plates; 
			6.5 mm, 0.4 &#956;M; 
			pore size</td>
		</tr>
	</tbody>
</table>

development of a human preclinical model of osteoclastogenesis from peripheral blood monocytes co-cultured with breast cancer cell lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

A method was optimized to easily differentiate osteoclasts from human peripheral blood monocytes. The monocyte culture was cultured with cancer cells, confirming (as described in the literature18) that cancer cells are capable of sustaining osteoclastogenesis in bone metastases. Osteoclasts differentiated from cancer cells and GFs are shown in Figure 1. An osteoclast-like cell is a cell with 4 or more nuclei and is positive for TRAP st...

Watch this Scientific Journal Video about Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines at JoVE.com

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

The overall goal of this in vitro human preclinical model of osteoclastogenesis derived from peripheral blood monocytes, or PBMCs, cultured with breast cancer cell lines is to mimic cancer cell osteoclast interactions. This method can help to answer key questions in the field of bone metastasis about the effects of cancer cell and bone cell interaction within the bone microenvironment. The main advantage of this technique is that it is a fully human pre-clinical model.Also, this method can provide insight into bone metastasis. It can also be applied to the study of other bone-related pathological mechanisms, such as osteoporosis. Individuals new to this method may struggle because of the variability among healthy donors and in selecting the appropriate PBMC cell density.Visual demonstration of this method is critical as they need see our PBMC selection and seeding steps require some manual experience before proficiency can be achieved. Begin by diluting at least 20 milliliters of healthy-donor whole blood in EDTA at a one to one ratio in PBS. After mixing thoroughly, split the blood solution into 30 milliliter aliquots in 50 milliliter conical tubes.Carefully underlay 15 milliliters of lymphocyte separation medium into the bottom of each tube. Separate the cells by density gradient centrifugation. And pull the white mononuclear-cell containing buffy coats into a new 15 milliliter tube.Wash the harvested cells two times in 20 milliliters of PBS per wash, followed by lysis of the red blood cells with five milliliters of red blood cell lysis buffer for three to five minutes, on ice. Stop the reaction with 20 milliliters of PBS, and collect the white blood cells by centrifugation. Resuspend the pellet and complete alpha M-E-M for counting.Then, plate the cells at a seven point five times 10 to the five PBMC per square centimeter concentration in each well of a 24 well plate for their incubation at 37 degrees celsius and five percent CO2. After about three hours, remove the supernatant, debris, unattached cells, and unlysed erythrocytes from the cultures. And add fresh medium, supplemented with MCSF.Fourteen days after seeding, wash the cells two times with PBS, and fix them in four percent paraformaldehyde for 20 minutes at room temperature. Perform trap staining according to the manufacturer's instructions. The osteoclast-like cells will be TRAP positive with at least four nuclei.Count the osteoclast-like cells manually under the microscope at a 10X magnification. For cancer cell induced osteoclast differentiation when the cancer cells reach ninety-percent confluency, they're detached by trypsinization and seeded onto zero point four micrometer pore inserts at a four times 10 to the three cells per square centimeter concentration in fresh alpha MEM. The next day, place the seeded inserts over undifferentiated one-day plated mononuclear cell cultures in complete alpha M-E-M, and change the medium every two to three days.Then, 11 days after the start of the co-culture, transfer the inserts into a new plate containing the appropriate reagent for the desired downstream analysis. Breast cancer cells can sustain osteoclastogenesis, as the number of osteoclasts in the wells induced by cancer cells is similar to that obtained in the positive growth-factor control wells. The number of osteoclasts observed in all of the cultures is reduced however, when the cells are treated with an anti-tumor drug.Interestingly, the surface areas of matured osteoclasts derived from growth factors are larger than those induced by cancer cells. This effect is not observed in anti-tumor drug treated cultures. Once mastered, this technique can be completed in seven eight hours if it is performed properly.Following this procedure, additional downstream analysis can be performed as gene-expression analysis, Western Blot, immunofluorescence analysis, or ELISA to answer additional questions about cancer-cell/bone-cell interactions or about anti-tumor drug mechanisms. After its development this technique pave the way for researchers in the field of bone metastasis to study the bone microenvironment, in fully human preclinical model. After watching this video you should have a good understanding of how to co-culture monocytes, undergo differentiation with cancer cells.Don't forget that working with biological samples and drugs can be extremely hazardous and that precautions such as wearing gloves and lab coats should always be taken while performing this procedure.

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Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

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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 ...

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Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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 ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...