This work was supported by National Natural Science Foundation of China 81402582, Natural Science Foundation of Shanghai 12DZ2295100, 14YF1400600 and 18ZR1404500

All animal procedures were approved and followed the guidelines of the Animal Ethics Committee at Fudan University and all pancreatic cancer specimens were obtained from Fudan University Shanghai Cancer Center. Ethical approval was obtained from the FUSCC Ethics Committee, and written informed consent was obtained from each patient.
1. Preparing the Equipment for the Microinjection
<ol>
	<li>Preparing the injection plate.
	<ol>
		<li>Prepare a 50 mL solution of 1% agarose di...

<ol>
	<li>Collins, D. C., Sundar, R., Lim, J. S. J., Yap, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+Precision+Medicine+in+the+Clinic:+From+Biomarker+Discovery+to+Novel+Therapeutics.">Towards Precision Medicine in the Clinic: From Biomarker Discovery to Novel Therapeutics.</a> Trends in Pharmacological Sciences. 38 (1), 25-40 (2017).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+pancreatic+cancer+modeling+and+drug+screening+using+human+pluripotent+stem+cell-+and+patient-derived+tumor+organoids.">Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.</a> Nature Medicine. 21 (11), 1364-1371 (2015).</li><li>Pauli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+In+Vitro+and+In+Vivo+Cancer+Models+to+Guide+Precision+Medicine.">Personalized In Vitro and In Vivo Cancer Models to Guide Precision Medicine.</a> Cancer Discovery. 7 (5), 462-477 (2017).</li><li>Hidalgo, 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4 (9), 998-1013 (2014).</li><li>Jung, J., Seol, H. S., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Generation+and+Application+of+Patient-Derived+Xenograft+Model+for+Cancer+Research.">The Generation and Application of Patient-Derived Xenograft Model for Cancer Research.</a> Cancer Research and Treatment. 50 (1), 1-10 (2018).</li><li>Fior, R., 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=Single-cell+functional+and+chemosensitive+profiling+of+combinatorial+colorectal+therapy+in+zebrafish+xenografts.">Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (39), E8234-E8243 (2017).</li><li>Chen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+xenograft+model+for+studying+human+cancer+stem+cells+in+distant+metastasis+and+therapy+response.">A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response.</a> Methods in Cell Biology. 138, 471-496 (2017).</li><li>Gaudenzi, G., 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=Patient-derived+xenograft+in+zebrafish+embryos:+a+new+platform+for+translational+research+in+neuroendocrine+tumors.">Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors.</a> Endocrine. 57 (2), 214-219 (2017).</li><li>Lee, J. Y., Mazumder, A., Diederich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Assessment+of+the+Bioactivity+of+the+Anticancer+Coumarin+OT48+by+Spheroids,+Colony+Formation+Assays,+and+Zebrafish+Xenografts.">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a> Journal of Visualized Experiment. (136), (2018).</li><li>Zhang, 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=Adipocyte-Derived+Lipids+Mediate+Melanoma+Progression+via+FATP+Proteins.">Adipocyte-Derived Lipids Mediate Melanoma Progression via FATP Proteins.</a> Cancer Discovery. 8 (8), 1006-1025 (2018).</li><li>Howe, 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nature Reviews: Genetics. 8 (5), 353-367 (2007).</li><li>Guo, 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=U0126+inhibits+pancreatic+cancer+progression+via+the+KRAS+signaling+pathway+in+a+zebrafish+xenotransplantation+model.">U0126 inhibits pancreatic cancer progression via the KRAS signaling pathway in a zebrafish xenotransplantation model.</a> Oncology Reports. 34 (2), 699-706 (2015).</li><li>Yao, 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=Canonical+Wnt+Signaling+Remodels+Lipid+Metabolism+in+Zebrafish+Hepatocytes+following+Ras+Oncogenic+Insult.">Canonical Wnt Signaling Remodels Lipid Metabolism in Zebrafish Hepatocytes following Ras Oncogenic Insult.</a> Cancer Research. 78 (19), 5548-5560 (2018).</li><li>Veinotte, C. J., Dellaire, G., Berman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooking+the+big+one:+the+potential+of+zebrafish+xenotransplantation+to+reform+cancer+drug+screening+in+the+genomic+era.">Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era.</a> Disease Models &amp; Mechanisms. 7 (7), 745-754 (2014).</li><li>Zon, L. I., Peterson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+age+of+chemical+screening+in+zebrafish.">The new age of chemical screening in zebrafish.</a> Zebrafish. 7 (1), 1 (2010).</li><li>Lam, S. H., Chua, H. L., Gong, Z., Lam, T. J., Sin, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+maturation+of+the+immune+system+in+zebrafish,+Danio+rerio:+a+gene+expression+profiling,+in+situ+hybridization+and+immunological+study.">Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.</a> Developmental &amp; Comparative Immunology. 28 (1), 9-28 (2004).</li><li>Mercatali, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Patient-Derived+Xenograft+(PDX)+of+Breast+Cancer+Bone+Metastasis+in+a+Zebrafish+Model.">Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model.</a> International Journal of Molecular Sciences. 17 (8), (2016).</li><li>Wu, J. Q., 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=Patient-derived+xenograft+in+zebrafish+embryos:+a+new+platform+for+translational+research+in+gastric+cancer.">Patient-derived xenograft in zebrafish embryos: a new platform for translational research in gastric cancer.</a> Journal of Experimental and Clinical Cancer Research. 36 (1), 160 (2017).</li><li>Tulotta, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Cancer+Angiogenesis+and+Metastasis+in+a+Zebrafish+Embryo+Model.">Imaging Cancer Angiogenesis and Metastasis in a Zebrafish Embryo Model.</a> Advances in Experimental Medicine and Biology. 916, 239-263 (2016).</li><li>Yao, 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=Screening+in+larval+zebrafish+reveals+tissue-specific+distributions+of+fifteen+fluorescent+compounds.">Screening in larval zebrafish reveals tissue-specific distributions of fifteen fluorescent compounds.</a> Disease Model&amp; Mechanisms. , 028811 (2017).</li><li>Tentler, J. J., 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nature Reviews: Clinical Oncology. 9 (6), 338-350 (2012).</li><li>Charo, J., 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=Bcl-2+overexpression+enhances+tumor-specific+T-cell+survival.">Bcl-2 overexpression enhances tumor-specific T-cell survival.</a> Cancer Research. 65 (5), 2001-2008 (2005).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+contribute+to+embryonic+and+extraembryonic+lineages+in+mouse+embryos+upon+inhibition+of+apoptosis.">Human embryonic stem cells contribute to embryonic and extraembryonic lineages in mouse embryos upon inhibition of apoptosis.</a> Cell Research. 28 (1), 126-129 (2018).</li><li>Boise, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bcl-x,+a+bcl-2-related+gene+that+functions+as+a+dominant+regulator+of+apoptotic+cell+death.">bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.</a> Cell. 74 (4), 597-608 (1993).</li><li>Moore, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+imaging+of+normal+and+malignant+cell+engraftment+into+optically+clear+prkdc-null+SCID+zebrafish.">Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.</a> Journal of Experimental Medicine. 213 (12), 2575-2589 (2016).</li></ol>

Both PDX and CDX models are vital platforms in the field of tumor biology22, and the critical step of a successful inter-species transplantation is to improve the survival of the xenograft. &#160;Recently, some studies have shown that transient expression of BCL2L1 (BCL-XL) or BCL2 may significantly improve the viability of human embryonic stem cells in mice hosts without affecting the cell identities and fates23,24</s...

Precision oncology aims to find the most beneficial therapeutic strategies for individual patient1. Currently, numerous preclinical models such as in vitro primary culture, in vitro organoid culture2, and patient-derived xenografts (PDX) in mice before or after organoid culture are proposed for diagnosis and to screen/assess the potential therapeutic choices3. PDX model generated by the injection of human primary cancer cells into immune-compromised mice, is one of the most promising tools for personalized drug screening in clinical oncology3,4. Unlike the cultured cell line in vitro, PDX models usually preserve the integrity and heterogeneity of the in vivo tumor environment, better mimicking the diversity and idiosyncratic characteristics of different tumor patients, and therefore, may predict the potential medical outcome of patients4. However, the generation of PDX models in mice requires high quality patient samples and months of time to gather sufficient cells and models for multi group experiments, and the cellular/genetic compositions of the xenograft may drift from those of the original patient&#8217;s biopsy. The success rate for establishing mice PDX model is also low, making it difficult to be broadly implemented in clinical practice. For the patients carrying rapidly progressed cancers like pancreatic cancer, they may not be able to obtain valuable information from the PDX experiments in time.&#160;
In the past few years, zebrafish has been reported to be potential hosts for not only CDX (cell-derived tumor xenograft) models, but also PDX models5,6,7,8,9,10. As a vertebrate model animal, zebrafish harbors sufficient similarities with mammals in both genetics and physiology, with two significant advantages: transparency and small in size11. Zebrafish is also highly fecundity, and hundreds of inbred larvae can be obtained within a few days from a single pair of adults12. Several studies have employed zebrafish to generate both transgenic and xenograft models of cancer diseases13,14. Compared to mice xenografts, zebrafish xenografts allow tracking at single cell resolution. A certain amount of human tissues is capable of generating hundreds of zebrafish PDX models (zPDXs), while may only be sufficient to generate a couple of mice PDX models15,16. Besides, the zebrafish larvae at 2-5 dpf already develop complete circulatory systems and metabolic organs such as liver and kidney, but not the immune system17, while the remaining yolk sac is a natural 3D medium, ideal for drug screening, drug resistance tests and tumor migration observations6,18,19,20,21.
With an ultimate attempt to use zPDX as a screening/testing platform for clinical use, here, we describe an optimized proposal for zPDX model of pancreatic cancer, which allows the in vivo candidate drug assessment within a short time using fewer cells at lower costs. Compared to the previous references about zPDX6,9,10, we introduced several optimizations to make the system more feasible and reliable for clinical personalized diagnosis: 1) pre-sorting different cell groups in the primary tumor tissues and stabilizing primary cells for one week before further experiments; 2) labeling the human cells and enhancing the cell viability in xenograft via lentivirus-based genetic modification; 3) optimizing the zebrafish culture condition in both nutriment supplements (glucose and glutamine) and temperature; 4) quantifying the drug responses of different cell types in a comparative manner. We also made changes to the injection solution by adding several supplementary materials. Altogether, those improvements provide the possibility to quickly generate a more patient-like xenograft in zebrafish hosts that can be used as a reliable tool to assess the response of candidate drugs.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>sv30087.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Cliniscience</td>
			<td>Y0503</td>
			<td>Rho kinase inhibitor</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>invivogen</td>
			<td>ant-pm-1</td>
			<td>an antibiotic for primary cell cultures</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide&#160;</td>
			<td>Sigma</td>
			<td>N3376</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin streptomycin</td>
			<td>GIBCO</td>
			<td>15140122.00</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer (PBS)</td>
			<td>GIBCO</td>
			<td>C10010500CP</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS&#160;</td>
			<td>GIBCO</td>
			<td>14170112.00</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase type IV</td>
			<td>GIBCO</td>
			<td>17104019.00</td>
			<td></td>
		</tr>
		<tr>
			<td>hyaluronidase</td>
			<td>Sigma</td>
			<td>H3884</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase&#8544;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>b-FGF</td>
			<td>GIBCO</td>
			<td>PHG0264</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>GIBCO</td>
			<td>PHG0314</td>
			<td></td>
		</tr>
		<tr>
			<td>pancreatic cancer fibroblasts inhibitor</td>
			<td>CHI Scientific</td>
			<td>FibrOUT</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m sterile filter</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>concentration column</td>
			<td>Millipore</td>
			<td>Millipore UFC910008</td>
			<td>Concentrate the virus</td>
		</tr>
		<tr>
			<td>polybrene&#160;</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic Acid Sodium Salt</td>
			<td>Sigma</td>
			<td>H7630</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>GIBCO</td>
			<td>21051024.00</td>
			<td></td>
		</tr>
		<tr>
			<td>gemcitabine</td>
			<td>Gemzan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methylcellulose</td>
			<td>Sigma</td>
			<td>M0262</td>
			<td></td>
		</tr>
		<tr>
			<td>Navitoclax(ABT-263)</td>
			<td>Selleck</td>
			<td>S1001</td>
			<td>Bcl-xL inhibitor</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>NARISHIGE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>LEICA</td>
			<td>SP8</td>
			<td>0.00</td>
		</tr>
	</tbody>
</table>

patient-derived heterogeneous xenograft model of pancreatic cancer using zebrafish larvae as hosts for comparative drug assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

A schematized outline of the procedure is represented in Figure 1. In short, the primary cancer tissue cells were seeded into the complete medium after digestion with or without the addition of pancreatic cancer fibroblast inhibitors. Cancer cells and fibroblasts were enriched as two distinct populations that fibroblasts dominated without inhibitors, and cancer cell growth prevailed after the addition of inhibitors (Figure 2). Tw...

Watch this Scientific Journal Video about Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment at JoVE.com

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Today we're going to introduce a larvae Zebrafish PDX Model. This model is significant because they can better mimic a similar cellular composition in the xenograft for assessing drug responses and also further improves the consistency of the results with our comparative analysis. There are two main advantages of this technique.Firstly, we use various standard chemical dyes to label cells into different fluorescence, allowing the real-time tracing of the cellular compositions and the ability of the anagraph phase. Secondly, it also improves the general survival time of human cells in zebrafish, their genetic modification, which provides a longer observation window for drug testing. This technique provides a potential model for personalized medicine for patients with pancreatic cancer and also for the pre-assessment of pre-clinical tumor drug.This method can also be used to trace other tumor cells or no tumor cell xenografted in zebrafish in vivo and to improve the cell survival for longer observation time. To prepare the injection plate, prepare a 50 milliliter solution of 1%agarose in E3 solution. Boil the solution until the agarose dissolves.Pour the entire solution into a 10 centimeter Petri dish and then place the Zebrafish embryo fixation mold onto the surface. When the agarose solution solidifies completely, remove the mold. To prepare injection needles, use a needle puller to pull a 10 centimeter glass capillary with an inner dimension of 0.9 millimeters into two needles.Using a microscope and forceps, cut the end of the needle to create an opening. First, place one to two pairs of adult Zebrafish into a mating tank around seven to nine PM.At eight AM the next morning, collect the fertilized eggs. Transfer the fertilized eggs to a Petri dish containing 40 milliliters of fresh E3 solution.Incubate at 28.5 degrees Celsius. First, transfer the collected pancreatic cancer sample into a Petri dish. Rinse the tissue five to six times with PBS and use a scalpel to cut the tissue into one millimeter cubed pieces.Next, transfer the shredded tissue into a 50 milliliter tube containing five milliliters of HBSS. Add collagenase type four, hyalurodinase, and DNAse one at the final concentrations shown here. Pipette the mixture up and down to mix well.Incubate the mixture at 37 degrees Celsius in a 5%carbon dioxide incubator for 15 to 20 minutes. Pipette the mixture up and down a few times every five minutes. Add seven milliliters of DMEM to the tube.Centrifuge at 110 times G and at four degrees Celsius for five minutes. Then, decant the supernatant and re-suspend the tumor mixture in DMEM. Plate the mixture into two six centimeter Petri dishes containing three milliliters of full growth media named group one and group two.Incubate both groups at 37 degrees Celsius in a 5%carbon dioxide incubator. After 48 hours, add 100x inhibitor of pancreatic cancer fibroblasts into the medium of group one to remove the overgrown fibroblasts, leaving the cancer cells as the major cell types. Continue incubating using the previous conditions.After producing the lentivirus, seed the cells to be infected into a 12-well plate with 30 to 40%density. Culture the cells overnight at 37 degrees Celsius in a 5%carbon dioxide incubator. The next day, replace the medium with 500 microliters of serum-free medium containing polybrene at a concentration of eight micrograms per milliliter.Incubate at 37 degrees Celsius with 5%carbon dioxide for four hours. Then, add an additional 100 microliters of the lentivirus to the medium and incubate again for 12 hours. After 12 hours, replace the medium with two milliliters of complete medium and incubate for an additional 36 hours.After 36 hours, check the fluorescence markers. Harvest the infected cells and mix them at a one-to-one ratio with a final concentration of one million cells per milliliter. Centrifuge the cells at 110 times G for five minutes and re-suspend the cell mixture in 50 microliters of injection solution.After anesthetizing the Zebrafish larvae, transfer them into the injection plate, which is filled with modified E3.Take up 25 microliters of the mixed cell suspension into the microcapillary needle and insert the needle into the microinjection manipulator. Set the injection pressure and time and inject 50 to 80 cells into the yolk sac of 48 hours post-fertilization Zebrafish. First, transfer the post-zenografted Zebrafish larvae into 40 milliliters of mix solution at 32 degrees Celsius.Place 10 zenografted larvae into each well of a 12-well plate. Next, divide the larvae into four groups. Treat the control group with E3 containing 0.1%DMSO and treat the other groups with Gemcitabine and/or Navitoclax.Incubate all larvae at 32 degrees Celsius for two days. After anesthetizing the zenografted larvae post-treatment, place them in 3%methyl cellulose. Use a fluorescence or confocal microscope to image the larvae from the lateral view.Then, use image J and graph pad software to quantify the intensity of red and green fluorescence signals. In this study, primary cancer tissue cells are seeded into complete medium after digestion with or without the addition of pancreatic cancer fibroblasts inhibitors. Cancer cells and fibroblasts are enriched as two distinct populations.The population without inhibitors is dominated by fibroblasts while cancer cell growth prevails after the addition of inhibitors. Two lentiviral packaging vectors are constructed which express green or red fluorescent proteins in BCL2L1. The virus-based fluorescence labeling represents the survival status of the cells.The mixed cancer cells and fibroblasts are injected into the Zebrafish yolk sac at 48 hours post-fertilization and are then treated with Gemcitabine and/or Navitoclax for two days. The cell viabilities in different populations and cellular composition of the zenograft are changed as the responses to the drug treatment. When processing different types of tumors, there are several steps that may need to be objected, including cell digestion time, drug treatment dosage, and so on.This technique helps researchers to employ Zebrafish from a standard CGX or PDS assay. It allows for the mixing and injection of different cells at fixed ratios to quantify the progress bands by comparing them to cell growth. Personal protection is of great significance, especially when packaging lentivirus.Wear masks and gloves when appropriate and try not to mix on skin.

patient-derived-heterogeneous-xenograft-model-pancreatic-cancer-using

Research

JoVE Journal

Cancer Research

9.0K visualizações.  Fudan University. Hoje vamos introduzir um modelo PDX de zebrafish larvas. Este modelo é significativo porque eles podem imitar melhor uma composição celular semelhante no xenoenxerto para avaliar as respostas medicamentosas e também melhora ainda mais a consistência dos resultados com nossa análise comparativa. Há duas principais vantagens dessa técnica.Em primeiro lugar, usamos vários corantes químicos padrão para rotular as células em diferentes fluorescências, permitindo o rastreamento ...