This work was supported in part by the NIH R01CA221771 grant to A.M. and by the P41GM135018 grant to T.O. supporting the Quantitative Bio-Element Analysis and Mapping (QBEAM) Center at Michigan State University. We would like to thank Danielle Ferguson, DVM, MS, of the Department of Campus Animal Resources (CAR) at Michigan State University for supervising animal procedures and ensuring compliance with IACUC protocols and Nazanin Talebloo, PhD, for assistance with ICP-OES.

The Michigan State University Institutional Animal Care and Use Committee (IACUC) has approved all procedures involving animal subjects. Values for calculations are summarized in Table 1.
1. Key steps of MN-anti-miR10b synthesis
NOTE: Details of the MN-anti-miR10b synthesis have been described previously9,10,11.
<ol>
	<li>Prepare the magnetic nanoparticle (MN) core by co-precipitation method.</li>
	<li>Crosslink and aminate the prepared nanoparticles using sodium hydroxide, epichlorohydrin, and ammonium hydroxide.</li>
	<li>Conjugate a Cy5.5-NHS ester to MN through the heterobifunctional cross-linker N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP) to obtain MN-Cy5.5 to enable fluorescence imaging and microscopy.</li>
	<li>Activate anti-miR-10b locked nucleic acid with 3% tris(2-carboxyethyl)phosphine (TCEP) and conjugate to MN-Cy5.5 to yield the MN-anti-miR10b.</li>
	<li>Perform characterization of the conjugate to determine iron content (by iron assay), the number of Cy5.5 molecules per nanoparticle (by spectrophotometry) and the amount of conjugated LNA (by agarose gel electrophoresis).</li>
</ol>
2. Acquire study animals
<ol>
	<li>Outline the study in advance to plan the experimental groups and the number of animals per group. House up to 5 mice per cage. If multiple cages of 5 mice are used during treatment, be sure to have control and experimental mice within each cage to remove the cage as a confounding variable.</li>
	<li>Obtain mice at 6-7 weeks of age. Allow for at least 1 week of acclimation to the housing conditions prior to the induction of orthotopic tumors. 
	NOTE: Procedures using the MDA-MB-231 (human-derived cell line) model of spontaneous breast cancer metastasis are described below. For this model, athymic nude mice (Foxn1nu/Foxn1nu) are commonly used. Other compatible immunocompromised mouse strains may be used, and the procedures are also applicable to allograft models in immunocompetent mice (e.g., 4T1 breast cancer cells in BALB/c mice).</li>
</ol>
3. Culture cells
<ol>
	<li>Supplement Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (complete growth medium) to grow MDA-MB-231 cells expressing luciferase (e.g., MDA-MB-231-luc-D3H2LN). Grow the cells under aseptic conditions at 37 &#176;C with 5% CO2 and 95% humidity, generally in a T75 flask. If frozen, thaw cells and passage at least once prior to tumor implantation. Record the passage number on the flask. 
	NOTE: In this study, a previously frozen vial of 1 &#215; 106 cells was thawed and passaged twice before use.
	<ol>
		<li>To passage, apply 0.25% trypsin for 3 min at 37 &#176;C to trypsinize cells at a confluency of &#60;80%. Add 4x volume of complete growth media to neutralize the trypsin.</li>
		<li>Centrifuge the suspension at 200 &#215; g for 5 min in a conical tube. Resuspend the cell pellet in 5 mL of complete growth medium after decanting the supernatant. Aliquot a portion of the cells to a new flask and add additional complete growth medium based on the new flask volume. Update the passage number on the new flask.</li>
	</ol>
	</li>
	<li>Thaw new cells after 10 passages to minimize genetic drift across studies.</li>
</ol>
4. Preparation of cells for induction of orthotopic tumors
<ol>
	<li>Place frozen Matrigel (basement membrane matrix extract) at 4 &#176;C for 24 h prior to preparation to allow the matrix extract to liquefy.</li>
	<li>Determine the concentration and volume of cells needed for the study. Implant the mice with 1 &#215; 106 cells per mouse in a 50 &#181;L volume, composed of 1 part of chilled PBS and 1 part of the matrix extract.</li>
	<li>Pellet the trypsinized cells as described in step 3.1.2. Resuspend the cell pellet in at least 10 mL of PBS to wash the cells, then centrifuge once more at 200 &#215; g for 5 min. Resuspend the pellet in 500 &#181;L of chilled PBS (cell stock).</li>
	<li>Count the cells using a hemocytometer. As the cells will be at a high concentration, dilute a small aliquot (e.g., 10 &#181;L) as needed, keeping track of the dilution factor until accurate measurements can be taken.</li>
	<li>Dilute the required total number of cells to 40 &#215; 106 cells/mL, then add an equal volume of the chilled matrix extract to achieve a final concentration of 1 &#215; 106 cells per 50 &#181;L. Keep on ice to prevent the extract from solidifying prior to implantation. 
	NOTE: Cell concentrations are summarized in Table 1.</li>
</ol>
5. Induction of orthotopic tumors
<ol>
	<li>Anesthetize a mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0-3% isoflurane on a heating pad. Confirm the surgical plane of anesthesia by lack of corneal reflex and/or toe pinch response. Protect against corneal drying by applying ophthalmic ointment to the eyes.</li>
	<li>Clean the skin near the injection site with a 70% alcohol wipe and allow a few seconds for the alcohol to dry. Induce at mammary gland #4 to reduce the risk of overlap of bioluminescence imaging signals between the primary tumor and most common sites of metastasis (lung and axillary lymph node). 
	NOTE: Mammary gland numbering has been described previously12. Briefly, a mouse in supine position with head oriented upwards will have glands 1 to 5 on the injector&#39;s right side (mouse&#39;s left) beginning from the closest to the head (cervical - gland 1) and descending to the most caudal (inguinal - gland 5). Glands 6 to 10 are oriented similarly on the opposite side of the animal.</li>
	<li>Pipet the cell stock up and down to resuspend the cells. Draw 50 &#181;L of the ice-cold cell suspension into an insulin syringe with a 29 G needle and inject the cells immediately. Keep the syringe on ice if not able to inject immediately. 
	NOTE: Pipet up and down between each draw of cells to prevent the cells from settling.</li>
	<li>Insert the bevel directly below the nipple of the desired mammary gland parallel to the body of the mouse at that location and inject the cells at a steady, slow rate. Leave the needle within the skin for at least 5 s after completion of the injection to allow the Matrigel to solidify and prevent leakage.</li>
	<li>Move the mouse to a clean cage on a warming pad for recovery and supervise until fully ambulatory and able to maintain sternal recumbency. Do not leave the mouse unattended. Return the mouse to its cage with other mice only after it has recovered.</li>
</ol>
6. Monitoring tumor growth and metastasis development with bioluminescence imaging (BLI)
NOTE: As the MDA-MB-231 cells utilized here express luciferase, injection of luciferin substrate into mice will produce an optical signal detected by the imaging system scanner. In this model, metastases can be expected at 5-7 weeks post tumor induction. It is recommended to image mice 1-3x per week, depending on the importance of identifying the exact moment when metastases are visualized.
<ol>
	<li>Anesthetize the mice using 2% isoflurane to minimize the risk of injury to the mouse when administering luciferin. Protect against corneal drying by applying ophthalmic ointment to the eyes.</li>
	<li>Inject 150 mg/kg body weight of luciferin intraperitoneally into each mouse and return the mice to their cage on a warming pad to allow the mice to awaken and metabolize luciferin. Supervise and do not leave mice unattended until fully ambulatory and able to maintain sternal recumbency. 
	NOTE: Values used for dosing calculations are summarized in Table 1.</li>
	<li>Image the mice using the imaging system scanner beginning at approximately 10 min post injection with luciferin, re-anesthetizing the mice when needed to allow for time for transfer to the IVIS.
	<ol>
		<li>Image up to 5 mice together in supine position, taking care to ensure their entire bodies are included in the field of view guide markings and oriented as straight as possible. Use clear tape to secure their arms for better visualization of the axillary lymph nodes. If imaging several mice at once, use manifold dividers to separate the mice to prevent signals from radiating onto other mice. If dividers are not available, use strips of light-absorbing paper in their place (e.g., thick, black cardstock). 
		NOTE: Rates of luciferin metabolism vary across mouse and cell line models, and image acquisition beginning at 10 min post injection may not yield the strongest signal. It is recommended that, at the start of a new study, acquisitions at different time points be performed to determine the timing for peak signal intensity.</li>
		<li>Prepare the imaging system software for image acquisition with the following settings for BLI: Exposure = Auto, Binning = Medium, FStop = 1, Excitation = Block, Emission = Open, FOV = D, Height = 1.50.</li>
		<li>Generally, primary tumor signals will produce a relatively strong signal due to their superficial location using the setting Exposure = Auto. If monitoring for metastases, use black electrical tape to carefully cover the primary tumor and manually set Exposure = 300 s (or more) to acquire faint signals if present. 
		NOTE: In this model, a signal separate from the primary tumor that is visible over multiple imaging sessions when the lower threshold is set as 5 &#215; 103 radiance is considered indicative of metastasis.</li>
	</ol>
	</li>
</ol>
7. Resection of primary tumors
NOTE: Resection of primary tumors is important for longitudinal (e.g., therapeutic) studies in metastases; otherwise, mice may succumb to morbidity related to unrestricted primary tumor growth. Consider primary tumor size (risk of blood loss on resection) and ulceration (risk of infection) when determining time of resection.
<ol>
	<li>If considering the absence of BLI signal to test for successful primary tumor resection, perform pre-operation imaging as described in section 6&#160;and confirm that there is no signal post-surgery. Confirm successful resection within the next 1-2 days using freshly administered luciferin. 
	NOTE: Immediate re-administration of luciferin is not recommended to avoid unnecessary stress to the animal. Luciferin injected prior to primary tumor resection should be enough to detect the signal if the resection was not complete.</li>
	<li>Anesthetize the mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0-3% isoflurane on a heating pad. Confirm surgical plane of anesthesia by the lack of corneal reflex and/or toe pinch response.</li>
	<li>Protect against corneal drying by applying ophthalmic ointment to the eyes</li>
	<li>Inject 5 mg/kg ketoprofen subcutaneously as analgesia for the procedure. 
	NOTE: Values used for dosing calculations are summarized in Table 1.</li>
	<li>Prepare the surgical area by alternating scrubs of 70% alcohol and betadine 3x. Allow the final betadine scrub to dry before proceeding.</li>
	<li>Use sterile surgical scissors to open the skin above the primary tumor vertically (rostral-caudal). In the case of ulcerated skin, begin opening to the side of the ulceration to avoid leaving the primary tumor behind and to completely remove the ulcerated skin.</li>
	<li>Continue using scissors and forceps to carefully dissect the connective tissue around the encapsulated tumor to completely remove the mass from the skin, as well as underlying body tissues as any remaining tumor may regrow.</li>
	<li>If present, control bleeding by applying pressure with a cotton-tipped applicator.</li>
	<li>Close the surgical opening with 5-0 Vicryl suture. 
	NOTE: Interrupted suturing may result in better wound patency as mice are prone to bothering the surgical site.</li>
	<li>Allow the animal to recover in a clean cage on a warming pad until fully ambulatory. Place moistened food on the bottom of the home cage when returning the animal to the cage.</li>
	<li>Inject 5 mg/kg ketoprofen subcutaneously once per day for at least 2 days post surgery. Check wound health at these times.</li>
</ol>
8. Delivery of nanodrug
<ol>
	<li>Weigh the mice as nanodrug dosage is based on bodyweight.</li>
	<li>Anesthetize the mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0-3% isoflurane on a heating pad. Confirm surgical plane of anesthesia by the lack of corneal reflex and/or toe pinch response. 
	NOTE: Alternatively, the nanodrug can be administered to a restrained awake mouse. All subsequent steps would be the same.</li>
	<li>Prepare an insulin syringe with 29 G needle with 10 mg of Fe nanodrug/kg mouse body weight.</li>
	<li>Submerge the animal&#39;s tail in warm water (30-35 &#176;C) for 30 s to dilate the tail veins.</li>
	<li>Wipe excess water from the tail and clean the injection site with a 70% alcohol wipe.</li>
	<li>Insert the needle bevel up in the lateral tail vein approximately halfway down the tail and pull back the plunger slightly to confirm placement with flashback of blood into the needle. If required, move the needle slightly forward or to a more superficial depth to achieve successful placement.</li>
	<li>Upon successful insertion, inject the nanodrug steadily at a slow rate of approximately 5-10 s for a 40 &#956;L injection. Confirm successful injection by lack of solution pooling under the skin of the tail near the injection site and by darkening of the vein (from the dark nanoparticle solution). 
	NOTE: Values used for dosing calculations are summarized in Table 1. Clumped nanoparticle formulations may embolize to the lung. If respiratory distress is observed shortly after injection, gently perform chest compressions on the mouse. Immediate action always results in the successful recovery of the animal from this possible issue.</li>
	<li>Hold pressure over the injection site with gauze and remove the needle, keeping pressure for approximately 30 s until bleeding stops.</li>
	<li>Allow the animal to recover in a clean cage on a warming pad until fully ambulatory.</li>
</ol>
9. Collection of metastases for analysis
<ol>
	<li>Image the mice by BLI as described in section 6. 
	NOTE: In this study, 5 &#215; 103 radiance is considered indicative of metastasis and is generally observed at 5-7 weeks. A slight variance in time-to-metastasis is to be expected across mice.</li>
	<li>Immediately after imaging, sacrifice the mice by cervical dislocation under heavy (5%) isoflurane. Prior to dissection, confirm death by lack of corneal reflex and/or toe pinch response.</li>
	<li>Carefully collect the metastases. Lymph node metastases present as an enlarged, encapsulated mass. Lung metastases will generally be distributed throughout the lung parenchyma; hence, collect the whole lung.</li>
	<li>Place the collected tissues in a Petri dish and image using the imaging system to confirm bioluminescence (indicating the presence of luciferase-expressing cancer cells) and fluorescence (indicating nanodrug accumulation). Use the same BLI acquisition settings as described in step 6.3.2. FLI acquisition settings are Exposure = Auto, Binning = Medium, FStop = 1, Excitation = 675 and Emission = 720 (default program for Cy5.5 dye), Lamp Level = High, FOV = D, Height 1.50. Image the mouse carcass to determine if there is remaining cancer tissue worth collecting.</li>
	<li>Rinse the cancer tissues in PBS.</li>
	<li>To collect tissues for microscopy or qRT-PCR, embed in OCT and store at -80 &#176;C until ready for processing.</li>
	<li>To collect tissues for inductively coupled plasma optical emission spectroscopy (ICP-OES), tare a scale using an empty 1.7 mL tube, place the tissue in the tube, and record its weight. Freeze the tissue and store at -80 &#176;C until ready for processing.</li>
</ol>
10. Validation of nanodrug delivery by fluorescence microscopy
<ol>
	<li>Cryosection the OCT-embedded fresh frozen samples onto microscopy slides at 10 &#181;m thickness. Adjust the chamber and specimen holder temperatures according to the tissue type. Settings between -20 &#176;C and -15 &#176;C are appropriate for both lung and lymph tissue.</li>
	<li>Fix the tissue sections onto slides by submerging the sections or whole slides in 4% paraformaldehyde solution for 15 min. Rinse carefully with PBS.</li>
	<li>Mount coverslips onto the slides. Use a medium with 4&#39;,6-diamidino-2-phenylindole (DAPI) for visualization of tissue architecture.</li>
	<li>Use a fluorescence microscope to examine sections for Cy5.5 (excitation 683 nm/emission 703 nm), indicating nanodrug delivery. Confirm that the signal is not background by using a negative control sample (tissue from a non-injected animal).</li>
</ol>
11. Validation of nanodrug delivery by inductively coupled plasma optical emission spectroscopy (ICP-OES)
<ol>
	<li>Transfer the samples stored at -80 &#176;C to a 15 mL conical tube and incubate it in an oven with the cap removed at 37 &#176;C to dry. 
	NOTE: This process took 24 h for MDA-MB-231 metastasis samples but may take several days depending on the size and moisture content of the sample.</li>
	<li>Record the dry weight using a balance.</li>
	<li>Add 2 mL of 70% trace HNO3 to the vessel and microwave digest the sample. The parameters used in this study are as follows: Power = 1030 - 1800 W, Ramp Time = 20:00 - 25:00, Hold Time = 15:00, Temperature = 200 &#176;C, Cooling = 30 min. 
	CAUTION: Exercise caution when working with nitric acid as it and the fumes generated when heating it are highly corrosive. Work should be completed in a well-ventilated space with full personal protective equipment, such as lab coat, goggles/face shield, and gloves compatible with acid work. The small volumes and lengthy cooling used here minimize risk somewhat, but care should always be taken.</li>
	<li>Transfer the digested samples to metal-free conical tubes; then, transfer 300 &#181;L to a new tube. Dilute to 10 mL using 9.7 mL of ultrapure water, resulting in an HNO3 concentration of 3% (v/v).</li>
	<li>Prepare individual element Fe standards at concentrations of 1000, 100, 10, 1, 0.1, and 0 &#181;g Fe/mL in 3% HNO3 (v/v) and ultrapure water. Prepare individual element Y internal standard at a concentration of 1 &#181;g/mL in 3% HNO3 (v/v) in ultrapure water.</li>
	<li>Analyze samples using ICP-OES. For both axial and radial modes, select the following emission lines for analysis of iron content. Fe (234.350 nm), Fe (238.204 nm), Fe (259.940 nm), and Y (371.029 nm) used for internal standardization.</li>
	<li>Normalize the results to the amount of sample used for the input to calculate &#181;g of Fe/g of tissue.</li>
</ol>

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

<ol><li>Houssami, N., Macaskill, P., Balleine, R. L., Bilous, M., Pegram, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Her2+discordance+between+primary+breast+cancer+and+its+paired+metastasis%3A+Tumor+biology+or+test+artefact%3F+Insights+through+meta-analysis%5BTitle%5D)+AND+%22Breast+Cancer+Res+Treat%22%5BJournal%5D)">Her2 discordance between primary breast cancer and its paired metastasis: Tumor biology or test artefact? Insights through meta-analysis.</a> Breast Cancer Res Treat. 129 (3), 659-674 (2011).</li>
<li>Ma, L., Teruya-Feldstein, J., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour+invasion+and+metastasis+initiated+by+microRNA-10b+in+breast+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Tumour invasion and metastasis initiated by microRNA-10b in breast cancer.</a> Nature. 449 (7163), 682-688 (2007).</li>
<li>Yoo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combining+mir-10b-targeted+nanotherapy+with+low-dose+doxorubicin+elicits+durable+regressions+of+metastatic+breast+cancer%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Combining mir-10b-targeted nanotherapy with low-dose doxorubicin elicits durable regressions of metastatic breast cancer.</a> Cancer Res. 75 (20), 4407-4415 (2015).</li>
<li>Baffa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+expression+profiling+of+human+metastatic+cancers+identifies+cancer+gene+targets%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets.</a> J Pathol. 219 (2), 214-221 (2009).</li>
<li>Yigit, M. V., Moore, A., Medarova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+nanoparticles+for+cancer+diagnosis+and+therapy%5BTitle%5D)+AND+%22Pharm+Res%22%5BJournal%5D)">Magnetic nanoparticles for cancer diagnosis and therapy.</a> Pharm Res. 29 (5), 1180-1188 (2012).</li>
<li>Kumar, M., Yigit, M., Dai, G., Moore, A., Medarova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-guided+breast+tumor+therapy+using+a+small+interfering+RNA+nanodrug%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Image-guided breast tumor therapy using a small interfering RNA nanodrug.</a> Cancer Res. 70 (19), 7553-7561 (2010).</li>
<li>Yigit, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Context-dependent+differences+in+mir-10b+breast+oncogenesis+can+be+targeted+for+the+prevention+and+arrest+of+lymph+node+metastasis%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Context-dependent differences in mir-10b breast oncogenesis can be targeted for the prevention and arrest of lymph node metastasis.</a> Oncogene. 32 (12), 1530-1538 (2013).</li>
<li>Yoo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapy+targeted+to+the+metastatic+niche+is+effective+in+a+model+of+stage+iv+breast+cancer%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Therapy targeted to the metastatic niche is effective in a model of stage iv breast cancer.</a> Sci Rep. 7, 45060(2017).</li>
<li>Savan, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Case+report%3A+MicroRNA-10b+as+a+therapeutic+target+in+feline+metastatic+mammary+carcinoma+and+its+implications+for+human+clinical+trials%5BTitle%5D)+AND+%22Front+Oncol%22%5BJournal%5D)">Case report: MicroRNA-10b as a therapeutic target in feline metastatic mammary carcinoma and its implications for human clinical trials.</a> Front Oncol. 12, 959630(2022).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-administration+of+temozolomide+%28tmz%29+and+the+experimental+therapeutic+targeting+mir-10b%2C+profoundly+affects+the+tumorigenic+phenotype+of+human+glioblastoma+cells%5BTitle%5D)+AND+%22Front+Mol+Biosci%22%5BJournal%5D)">Co-administration of temozolomide (tmz) and the experimental therapeutic targeting mir-10b, profoundly affects the tumorigenic phenotype of human glioblastoma cells.</a> Front Mol Biosci. 10, 1179343(2023).</li>
<li>Robertson, N., Wang, P., Talebloo, N., Yamada, K., Moore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+of+siRNA-conjugated+dextran-coated+iron+oxide+nanoparticles+for+islet+protection+during+transplantation+and+noninvasive+imaging%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Synthesis of siRNA-conjugated dextran-coated iron oxide nanoparticles for islet protection during transplantation and noninvasive imaging.</a> Methods Mol Biol. 2592, 163-174 (2023).</li>
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<li>Kumar, M., Medarova, Z., Pantazopoulos, P., Dai, G., Moore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+membrane-permeable+contrast+agent+for+brain+tumor+detection+by+MRI%5BTitle%5D)+AND+%22Magn+Reson+Med%22%5BJournal%5D)">Novel membrane-permeable contrast agent for brain tumor detection by MRI.</a> Magn Reson Med. 63 (3), 617-624 (2010).</li>
<li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrigel%3A+From+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Matrigel: From discovery and ECM mimicry to assays and models for cancer research.</a> Adv Drug Deliv Rev. 79-80, 3-18 (2014).</li>
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Z.M and A.M. are co-founders and shareholders of TransCode Therapeutics Inc.

Nanoparticles have great potential for cancer treatment. Here, we showed that a Cy5.5-conjugated MNP carrier can reach cancer tissues to deliver therapeutic oligonucleotides in a murine model of metastatic breast cancer. The ability to administer the nanodrug systemically while still achieving considerable accumulation in cancer tissues offers tremendous advantages over many existing ASO delivery methods, which commonly require local and often invasive administration. As target specificity is imperative to patient safety...

Despite many advances in the treatment of breast cancer, clinical options for metastatic disease remain limited. Patients commonly receive therapies targeted against drivers identified in the primary tumor, such as estrogen or HER2, but these drivers are not always conserved in metastases, rendering therapy ineffective1. Other systemic therapies, such as chemotherapy, are non-specific and known for their side effects. To develop effective options for the treatment of metastatic breast cancer, it is important to consider the biological drivers that allow cancer cells to spread and colonize distant sites. One of these drivers is miR-10b, an oncogenic microRNA, implicated in breast cancer cell viability, invasion, and migration, which has been shown to be sufficient to confer metastatic potential in otherwise-nonmetastatic breast cancer cells2,3. Importantly, miR-10b is also expressed at higher levels in metastases compared to matched primary tumors4, making it a promising target for the treatment of existing metastases.
Although miRNAs such as miR-10b have great potential as therapeutic targets for metastatic disease, the design of therapeutically viable methods for miRNA silencing presents unique challenges. Antisense oligonucleotides (ASOs) that bind their complementary miRNA sequence are commonly transferred to the cells in vitro using lipofection but cannot easily reach tumor cells in vivo due to inherent instability, risk of destruction by nucleases, short blood half-life, and the inability to enter cells due to charge-charge repulsion5. To combat these challenges, we developed a clinically applicable carrier for biomolecules using dextran-coated magnetic iron oxide nanoparticles (MNP)6. Amine groups on the nanoparticle allow for the conjugation of oligonucleotides, fluorescent dyes (e.g., Cy5.5), and targeting moieties. Additionally, the iron oxide core allows for in vivo monitoring of vehicle delivery using magnetic resonance imaging (MRI). We conjugated anti-miR-10b locked nucleic acid ASO and Cy5.5 to MNP to create a &#34;nanodrug&#34; referred to as MN-anti-miR10b, depicted in Figure 17.
In our previous studies, we showed that the nanodrug efficiently causes downregulation of miR-10b and inhibits the migration and invasion of triple-negative breast cancer cells in vitro7. In murine models of metastatic breast cancer, intravenous delivery of the nanodrug prevented the development of lymph node metastases or, if administered after lymph node metastasis formation, halted their growth7. Notably, the nanodrug was observed to readily accumulate in cancer tissues. While the nanodrug did not eradicate metastases on its own, in subsequent studies, we showed that combination treatment with adjuvant doxorubicin was curative in both immunocompromised and immunocompetent mouse models3,8. The effects of miR-10b inhibition by the nanodrug have&#160;also been seen in feline mammary carcinoma9.
To effectively treat breast cancer, it is imperative to demonstrate that the drug accumulates in tissues of interest. Here, we present a protocol for demonstrating the accumulation of the magnetic nanoparticle carrier used to deliver therapeutic anti-miR-10b ASOs to cancer tissues using multiple modalities in murine models of metastatic breast cancer.

<table><tbody><tr><td>Agilent 5800 ICP-OES</td><td>Agilent</td><td>5800 ICP-OES</td><td>For ICP-OES</td></tr><tr><td>Ammonium hydroxide</td><td>Thermo Fisher Scientific Inc</td><td>458680025</td><td>For nanodrug synthesis</td></tr><tr><td>Athymic nude &quot;J:NU&quot; mice</td><td>Jackson Laboratory</td><td>RRID:IMSR_JAX:007850</td><td>Immunocompromised mouse model</td></tr><tr><td>Betadine Surgical Scrub</td><td>Purdue</td><td>6761815101</td><td>For tumor resection</td></tr><tr><td>Cotton Tipped Applicators</td><td>Puritan</td><td>S-18991</td><td>For tumor resection</td></tr><tr><td>Crile Hemostats - Straight</td><td>F.S.T.</td><td>13004-14</td><td>For tumor resection</td></tr><tr><td>Cy5.5-NHS ester</td><td>Abcam</td><td>ab146455</td><td>For nanodrug synthesis</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>For cell culture of MDA-MB-231</td></tr><tr><td>Eclipse 50i Clinical Microscope</td><td>Nikon</td><td>50i-B</td><td>For imaging of cryosections</td></tr><tr><td>Epichlorohydrin</td><td>Thermo Fisher Scientific Inc</td><td>117780250</td><td>For nanodrug synthesis</td></tr><tr><td>Extra Fine Graefe Forceps</td><td>F.S.T.</td><td>11150-10</td><td>For tumor resection and metastasis dissection</td></tr><tr><td>Fe standard</td><td>Inorganic Ventures</td><td>CGFE1-500ML</td><td>For ICP-OES</td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>35-010-CV</td><td>For cell culture of MDA-MB-231</td></tr><tr><td>Fine Scissors - Sharp 10.5cm</td><td>F.S.T.</td><td>14060-10</td><td>For tumor resection and metastasis dissection</td></tr><tr><td>Flask (T-75)</td><td>Corning</td><td>430641U</td><td>For cell culture of MDA-MB-231</td></tr><tr><td>HNO3 nitric acid (70%, trace metal grade)</td><td>Fisher Chemical</td><td>A509P212</td><td>For ICP-OES</td></tr><tr><td>Insulin syringe 1 CC 29 G x 1/2&quot;</td><td>Becton, Dickinson</td><td>324704</td><td>For tumor implant</td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>11695067772</td><td>For mouse anesthetization</td></tr><tr><td>Isoflurane vaporizer</td><td>SOMNI Scientific</td><td>VS6002</td><td>For mouse anesthetization</td></tr><tr><td>Isopropyl alcohol (70%) wipe</td><td>Cardinal</td><td>MW-APL</td><td>For tumor resection</td></tr><tr><td>IVIS SpectrumCT In Vivo Imaging System</td><td>PerkinElmer/Revvity</td><td>128201</td><td>For bioluminescence imaging</td></tr><tr><td>IVISbrite D-Luciferin Potassium Salt</td><td>PerkinElmer/Revvity</td><td>122799-100MG</td><td>For bioluminescence imaging</td></tr><tr><td>Ketofen (ketoprofen)</td><td>Zoetis</td><td>10004031</td><td>For tumor resection</td></tr><tr><td>Leica CM1950</td><td>Leica</td><td>CM1950</td><td>For cryosectioning of OCT-embedded samples</td></tr><tr><td>MARS 6 microwave digestion system</td><td>CEM</td><td>MARS 6</td><td>For ICP-OES</td></tr><tr><td>Matrigel, growth factor-reduced</td><td>Corning</td><td>354230</td><td>For tumor implant of MDA-MB-231</td></tr><tr><td>MDA-MB-231-luc-D3H2LN</td><td>PerkinElmer/Revvity</td><td>119369</td><td>For mouse model of spontaneous metastasis</td></tr><tr><td>Metal-free polypropylene 15 mL conical tubes</td><td>Labcon</td><td>31343450019</td><td>For ICP-OES</td></tr><tr><td>Microcentrifuge tube (1.7 mL)</td><td>DOT Scientific</td><td>RN1700-GMT</td><td>For metastasis sample collection</td></tr><tr><td>N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP)</td><td>Thermo Fisher Scientific Inc</td><td>21857</td><td>For nanodrug synthesis</td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-144</td><td>For cell culture and tumor implant of MDA-MB-231</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td>For cell culture of MDA-MB-231</td></tr><tr><td>Puralube vet ointment</td><td>MWI Veterinary</td><td>27505</td><td>For tumor resection</td></tr><tr><td>Sodium hydroxide</td><td>Thermo Fisher Scientific Inc</td><td>3728-70</td><td>For nanodrug synthesis</td></tr><tr><td>Tissue-Tek Cryomold Intermediate 15 x 15 x 5 mm</td><td>Sakura</td><td>4566</td><td>For metastasis sample collection</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura</td><td>4583</td><td>For metastasis sample collection</td></tr><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Thermo Fisher Scientific Inc</td><td>T2556</td><td>For nanodrug synthesis</td></tr><tr><td>Trypsin, 0.25%</td><td>Gibco</td><td>25200-056</td><td>For cell culture of MDA-MB-231</td></tr><tr><td>Vicryl PLUS (Antibacterial) violet 27&quot; RB-1 taper</td><td>Ethicon</td><td>VCP303H</td><td>For tumor resection</td></tr></tbody></table>

monitoring of nanodrug accumulation in murine breast cancer metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

In our previous therapeutic in vivo studies, we treated mice with one dose of nanodrug (10 mg Fe nanodrug/kg mouse bodyweight) weekly for several weeks3,7,8. For this demonstration, we sought to determine whether accumulation of nanodrug could be observed in lung metastases after one dose, 1 week later. The results of this study would guide the timeline for monitoring the nanodrug accumulation in future longitudinal stu...

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

monitoring-of-nanodrug-accumulation-in-murine-breast-cancer-metastases

Research

JoVE Journal

Cancer Research

322 Views.  Michigan State University. Ons onderzoek richt zich op de behandeling van uitzaaiingen van borstkanker met behulp van beeldgeleide therapieën op een platform voor ijzeroxide-nanodeeltjes. Het doel is om effectieve therapieën te ontwikkelen die specifiek gericht zijn op uitgezaaide weefsels, de belangrijkste doodsoorzaken door kanker. Het kan een uitdaging zijn om te bepalen of systemische therapieën het gewenste weefsel hebben bereikt zonder dieren op te offeren.Ons nanodeelt...

Video: Monitoring van de accumulatie van nanodrugs bij borstkankermetastasen bij muizen

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Chemistry

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

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

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

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

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

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