Name: multimodal bioluminescent and positronic-emission tomography/computational tomography imaging of multiple myeloma bone marrow xenografts in nog mice
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Description: Watch this Scientific Journal Video about Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice at JoVE.c

This work was supported by a VA MERIT grant1I01BX001532 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (BLRDS) to P.F., and E.C. acknowledges support from the VA Clinical Science R&#38;D Service (Merit Award I01CX001388) and VA Rehabilitation R&#38;D Service (Merit Award I01RX002604). Further support came from a UCLA Faculty Seed Grant to J.K. These contents do not necessarily represent the views of the US Department of Veterans Affairs or the US Government.

All animal procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) of the Greater Los Angeles VA Healthcare system and were performed under sterile and pathogen-free conditions.
1.&#160;Preparation of Luciferase-expressing 8226 Cells (8226-LUC)
<ol>
	<li>Maintain the human MM cell line, 8226, in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in a humidified atmosphere containing 95% air ...

<ol>
	<li>Raab, M. S., Podar, K., Breitkreutz, I., Richardson, P. G., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Myeloma.">Multiple Myeloma.</a> The Lancet. 374 (9686), 324-339 (2009).</li><li>Galson, D. L., Silbermann, R., 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+Multiple+Myeloma+Bone+Disease.">Mechanisms of Multiple Myeloma Bone Disease.</a> BoneKEy Reports. 1, 135 (2012).</li><li>Anderson, K. C., Carrasco, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Myeloma.">Pathogenesis of Myeloma.</a> Annual Review of Pathology. 6, 249-274 (2011).</li><li>Reagan, M. R., Rosen, C. 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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=Laglambda-1:+A+Clinically+Relevant+Drug+Resistant+Human+Multiple+Myeloma+Tumor+Murine+Model+That+Enables+Rapid+Evaluation+of+Treatments+for+Multiple+Myeloma.">Laglambda-1: A Clinically Relevant Drug Resistant Human Multiple Myeloma Tumor Murine Model That Enables Rapid Evaluation of Treatments for Multiple Myeloma.</a> International Journal of Oncology. 28 (6), 1409-1417 (2006).</li><li>Miyakawa, Y., 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=Establishment+of+a+New+Model+of+Human+Multiple+Myeloma+Using+Nod/Scid/Gammac(Null)+(Nog)+Mice.">Establishment of a New Model of Human Multiple Myeloma Using Nod/Scid/Gammac(Null) (Nog) Mice.</a> Biochemical and Biophysical Research Communications. 313 (2), 258-262 (2004).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nod/Scid/Gamma(C)(Null)+Mouse:+An+Excellent+Recipient+Mouse+Model+for+Engraftment+of+Human+Cells.">Nod/Scid/Gamma(C)(Null) Mouse: An Excellent Recipient Mouse Model for Engraftment of Human Cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Frost, P., 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=In+Vivo+Antitumor+Effects+of+the+Mtor+Inhibitor+Cci-779+against+Human+Multiple+Myeloma+Cells+in+a+Xenograft+Model.">In Vivo Antitumor Effects of the Mtor Inhibitor Cci-779 against Human Multiple Myeloma Cells in a Xenograft Model.</a> Blood. 104 (13), 4181-4187 (2004).</li><li>Asosingh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Hypoxic+Bone+Marrow+Microenvironment+in+5t2mm+Murine+Myeloma+Tumor+Progression.">Role of the Hypoxic Bone Marrow Microenvironment in 5t2mm Murine Myeloma Tumor Progression.</a> Haematologica. 90 (6), 810-817 (2005).</li><li>Storti, P., 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=Hypoxia-Inducible+Factor+(Hif)-1alpha+Suppression+in+Myeloma+Cells+Blocks+Tumoral+Growth+in+Vivo+Inhibiting+Angiogenesis+and+Bone+Destruction.">Hypoxia-Inducible Factor (Hif)-1alpha Suppression in Myeloma Cells Blocks Tumoral Growth in Vivo Inhibiting Angiogenesis and Bone Destruction.</a> Leukemia. 27 (8), 1697-1706 (2013).</li><li>Fryer, R. 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Despite a variety of preclinical xenograft models of MM6,9,11,12,13, the ability to study the tumor/BM interactions within the BM microenvironment remains difficult14. The techniques described here allow for the rapid and reproducible engraftment of 8226-LUC tumors cells in the skeleton of NOG mice.
The criti...

MM is an incurable disease made up of malignant plasma B-cells that infiltrate the BM and cause bone destruction, anemia, renal impairment, and infection. MM makes up 10% - 15% of all hematological malignancies1 and is the most frequent cancer to involve the skeleton2. The development of MM stems from the oncogenic transformation of long-lived plasma cells that are established in the germinal centers of lymphoid tissues before eventually homing to the BM3. The BM is characterized by highly heterogeneous niches; including diverse and critical cellular components, regions of low pO2 (hypoxia), extensive vascularization, complex extracellular matrices, and cytokine and growth factor networks, all of which contribute to MM tumorgenesis4. Thus, the development of a disseminated MM xenograft model characterized by tumors that are strictly engrafted in the BM would be a very powerful and clinically relevant tool to study MM pathology in vivo5,6. However, numerous technical hurdles can limit the effectiveness of most xenograft models, making them costly and difficult to apply. This includes problems associated with consistent and reproducible tumor engraftment within the BM niche, a prolonged time to tumor development, and limitations in the ability to directly observe and measure changes of tumor growth/survival without having to sacrifice mice during the course of the experiment7,8.
This protocol uses a modified xenograft model that was initially developed by Miyakawa et al.9, in which an intravenous (IV) challenge with myeloma cells results in &#34;disseminated&#34; tumors that consistently and reproducibly engraft in the BM of NOD/SCID/IL-2&#947;(null) (NOG) mice10. The in situ visualization of these tumors is achieved by the stable transfection of the 8226 human MM cell line with a LUC allele and serially measuring the changes in the BLI produced by these engrafted tumor cells6. Importantly, this model can be expanded to utilize various other LUC-expressing human MM cell lines (e.g., U266 and OPM2) with a similar propensity to specifically engraft in the skeleton of NOG mice. The identification of the tumors by bioluminescent imaging of the mice is followed by measuring the uptake of radiopharmaceutical probes (such as 18F-FDG) by PET/CT. Together, this allows for additional characterization of critical biochemical pathways (i.e., alterations in metabolism, changes in hypoxia, and the induction of apoptosis) within the tumor/BM microenvironment. The major strengths of this model can be highlighted by the availability of a wide range of radiolabeled, bioluminescent and fluorescent probes and markers that can be used to study MM progression and pathology in vivo.

<table>
	<tbody>
		<tr>
			<td>8226 human myeloma cell line</td>
			<td>ATCC</td>
			<td>CCL-155</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ Mice (NOG)</td>
			<td>Jackson Labs</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGlo Luciferin substrate</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxyprobe-1Kit</td>
			<td>HPL</td>
			<td>HP1-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD45 (clone H130)</td>
			<td>BD Biosciences</td>
			<td>555483</td>
			<td>Used for flow cytometry to identify human CD45+ tumor cells in BM exudate</td>
		</tr>
		<tr>
			<td>rabbit anti-human CD45 (clone D3F8Q)</td>
			<td>Cell Signaling Technology</td>
			<td>70527</td>
			<td>Primary antibody used for Immunohistochemistry of excised bone</td>
		</tr>
		<tr>
			<td>Goat Anti-rabbit IgG (HRP conjugated)</td>
			<td>ABCAM</td>
			<td>ab205718</td>
			<td>Seconday antibody used for Immunohistochemistry of excised bone</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4.5 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E1310</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina XRMS In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sofie G8 PET/CT Imaging System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

multimodal bioluminescent and positronic-emission tomography/computational tomography imaging of multiple myeloma bone marrow xenografts in nog mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

In initial pilot studies, IV injections of 8226-LUC cells into NOD/SCID mice did not develop BM-engrafted MM tumors, although squamous MM tumors were easily formed (100% success rate). In contrast, IV challenges with 8226 cells into NOG mice generated (within 15 - 25 days) tumors in the skeleton (and only rarely formed tumors in non-skeletal tissue, such as the liver or spleen). Since tumors in the skeleton could not be visually confirmed by physically examining the animals, other methods...

Watch this Scientific Journal Video about Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice at JoVE.c

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

This method can be used to answer key questions about the role of the bone marrow micro-environment and the survival of engrafted myeloma tumors. The main advantage of this procedure is that it can be applied to longitudinal anti-drug studies and used to assay multiple biochemical pathways in a non-invasive manner. On the day of the experiment first wash the 8226 Luciferase transfacted cells three times in ice cold PBS at 200 RCF for five minutes per centrifigation and resuspend the pellet in fresh ice cold PBS at a five times ten to the sixth cells per 200 microliters of ice cold PBS per mouse.Next confirm a lack of response to toe pinch in an anesthetized four to six week old NOG mouse and apply ophthalmic ointment to the animal's eyes. Load the cells into a one milliliter insulin syringe equipped with a 26 gauge needle. Inject the entire volume of cells into the tail vein of the animal, before returning the mouse to it's cage, monitoring until full recovery.Ten to twenty days post challenge, inject each engrafted animal with 200 microliters of in vivo grade D-luciferin substrate in sterile saline intraperitonealy. Place the anesthetized animals in the supine position in a small animal imaging system within 5 to 10 minutes of the injection. Measure the average radiance in selected regions of interest within the corresponding imaging software.For targeted therapy of the 8226 Luciferase tumors, After measuring the baseline bioluminescence in each animal as just demonstrated, randomize the animals into treatment groups and treat each mouse with a series of 200 microliter IP injection of Temsirolimus or a saline control. Measure the Luciferase activity two times per week and plot the changes in bioluminescence over time. To measure changes in the tumor metabolism, first remove the food from the home cage of the mice for 24 hours to avoid excess nonspecific radiolabeled Fludeoxyglucose uptake.The next day dilute 500 to 100 microcuries of 18 Fluorine radiolabeled Fludeoxyglucose probe in sterile saline to a final volume of 100 microliters per mouse and record the time and activity of the probes with a dose calibrator. When the probes are ready place the first anesthetized animal on a heating pad with the head facing away and use a shielded one milliliter insulin syringe equipped with a 26 gauge needle to inject 100 microliters of the probe into the tail vein. Measure the residual radioactivity in the needle and syringe with the dose calibrator and note the activity and time.Then measure the radiolabeled Fludeoxyglucose activity in selected regions of interest that correspond to the engrafted tumors in a small animal PET CT imaging system. A successful bone marrow tumor engraftment can be confirmed by bioluminescence imaging as demonstrated. Serial imaging of multiple animals can be used to visualize the distribution of the bone marrow engrafted multiple myeloma tumors.Additionally optical imaging x-ray analysis shows a quick and non-invasive determination of the exact location and distribution of the multiple myeloma tumors within the mouse skeleton. The bioluminescence produced by these engrafted tumor cells can be serially and noninvasively measured to assess changes in tumor growth. Furthermore, the survival of mice treated with the mTOR inhibitor Temsirolimus can be monitored and positron emission and computed tomography analysis for tumor radiolabeled Fludeoxyglucose uptake can be used to demonstrate Temsirolimus mediated changes in glucose metabolism.While performing this procedure it's important to inject the cells IEV. We've found that failure to inject the cells into the vein results in the formation of non-marrow engrafted tumor cells near the site of injection. Following this procedure, other methods like immunohistochemistry and micro-CT can be used to address questions about the tumors impact on bone architecture and the non-tumor cellular and molecular components of the marrow environment.

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Cancer Research

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, ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as proteins, oligonucleotides, and synthetic small-molecule drugs. The SWCNT has customizable dimensions, a large superficial area, and can flexibly bind with drugs through different modifications on its surface; therefore, it is an ideal system to transport drugs into cells. Long noncoding RNAs (lncRNAs) are a cluster of noncoding RNA longer than 200 nt, which cannot be translated to protein but play an important role in biological and pathophysiological processes. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA. It was demonstrated that higher MALAT1 levels are related to the poor prognosis of various cancers, including multiple myeloma (MM). We have revealed that MALAT1 regulates DNA repair and cell death in MM; thus, MALAT1 can be considered as a therapeutic target for MM. However, the efficient delivery of the antisense oligo to inhibit/knockdown MALAT1 in vivo is still a problem. In this study, we modify the SWCNT with PEG-2000 and conjugate an anti-MALAT1 oligo to it, test the delivery of this compound in vitro, inject it intravenously into a disseminated MM mouse model, and observe a significant inhibition of MM progression, which indicates that SWCNT is an ideal delivery shuttle for anti-MALAT1 gapmer DNA.

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos

This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...