ai_agent

<meta charset="utf8"></head><body>患者来源的黑色素瘤类器官:癌症免疫治疗的前沿

<ol><li>Mirabelli, P., Coppola, L., Salvatore, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+Cell+Lines+Are+Useful+Model+Systems+for+Medical+Research%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">Cancer Cell Lines Are Useful Model Systems for Medical Research.</a> Cancers (Basel). 11 (8), 1098(2019).</li>
<li>Shannon, A. E., Boos, C. E., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-culturing+multicellular+tumor+models%3A+Modeling+the+tumor+microenvironment+and+analysis+techniques%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Co-culturing multicellular tumor models: Modeling the tumor microenvironment and analysis techniques.</a> Proteomics. 21 (9), e2000103(2021).</li>
<li>Jubelin, C., Muñoz-Garcia, J., Griscom, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+in+vitro+culture+models+in+oncology+research%5BTitle%5D)+AND+%22Cell+Biosci%22%5BJournal%5D)">Three-dimensional in vitro culture models in oncology research.</a> Cell Biosci. 12, 155(2022).</li>
<li>Jin, J., Yoshimura, K., Sewastjanow-Silva, M., Song, S., Ajani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+and+prospects+of+patient-derived+xenografts+for+cancer+research%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">Challenges and prospects of patient-derived xenografts for cancer research.</a> Cancers (Basel). 15 (17), 4352(2023).</li>
<li>Nabel, C. S., Heiden, M. G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+xenografts+to+study+cancer+metabolism%3A+when+does+X+mark+the+spot%29%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Patient-derived xenografts to study cancer metabolism: when does X mark the spot).</a> Cancer Res. 81 (17), 4399(2021).</li>
<li>Choi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+cancer+immunotherapy+using+patient-derived+xenografts+%28PDXs%29+in+humanized+mice%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.</a> Exp Mol Med. 50 (8), 99(2018).</li>
<li>Woo, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conservation+of+copy+number+profiles+during+engraftment+and+passaging+of+patient-derived+cancer+xenografts%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.</a> Nat Genet. 53 (1), 86(2021).</li>
<li>Somasundaram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor-infiltrating+mast+cells+are+associated+with+resistance+to+anti-PD-1+therapy%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.</a> Nat Commun. 12 (1), 346(2021).</li>
<li>Abdolahi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+xenograft+%28PDX%29+models%2C+applications+and+challenges+in+cancer+research%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Patient-derived xenograft (PDX) models, applications and challenges in cancer research.</a> J Transl Med. 20 (1), 206(2022).</li>
<li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoids%3A+The+current+status+and+biomedical+applications%5BTitle%5D)+AND+%22Med+Comm%22%5BJournal%5D)">Organoids: The current status and biomedical applications.</a> Med Comm. 4 (3), e274(2023).</li>
<li>Minoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bladder+cancer+organoids+as+a+functional+system+to+model+different+disease+stages+and+therapy+response%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Bladder cancer organoids as a functional system to model different disease stages and therapy response.</a> Nat Commun. 14, 2214(2023).</li>
<li>Ou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+melanoma+organoid+models+facilitate+the+assessment+of+immunotherapies%5BTitle%5D)+AND+%22eBioMedicine%22%5BJournal%5D)">Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.</a> eBioMedicine. 92, 104614(2023).</li>
<li>Grönholm, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+organoids+for+precision+cancer+immunotherapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Patient-derived organoids for precision cancer immunotherapy.</a> Cancer Res. 81 (12), 3149-3155 (2021).</li>
<li>Ou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+platforms+to+study+therapeutic+efficacy+of+human+%CE%B3%CE%B4+T+cells%5BTitle%5D)+AND+%22Clin+Transl+Med%22%5BJournal%5D)">Preclinical platforms to study therapeutic efficacy of human γδ T cells.</a> Clin Transl Med. 12 (6), e814(2022).</li>
<li>Murine L-Wrn conditioned medium protocol. The University of Texas MD Anderson Cancer Center. , Available from: 
 <a target="_blank" href="https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf">https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf</a> (2024).</li>
<li>Jee, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+collagen-based+3D+matrix+for+gastrointestinal+tract-derived+organoid+culture%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture.</a> Stem Cells Int. 13 (2019), 8472712(2019).</li>
<li>Pipkin, M. E., Rao, A., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transcriptional+control+of+the+perforin+locus%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">The transcriptional control of the perforin locus.</a> Immunol Rev. 235 (1), 55(2010).</li>
<li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tim-3+suppresses+the+killing+effect+of+V%CE%B39V%CE%B42+cells+on+colon+cancer+cells+by+reducing+perforin+and+granzyme+B+expression%5BTitle%5D)+AND+%22Exp+Cell+Res%22%5BJournal%5D)">Tim-3 suppresses the killing effect of Vγ9Vδ2 cells on colon cancer cells by reducing perforin and granzyme B expression.</a> Exp Cell Res. 386 (1), 111719(2020).</li>
<li>Li, L. T., Jiang, G., Chen, Q., Zheng, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ki67+is+a+promising+molecular+target+in+the+diagnosis+of+cancer+%28Review%29%5BTitle%5D)+AND+%22Mol+Med+Rep%22%5BJournal%5D)">Ki67 is a promising molecular target in the diagnosis of cancer (Review).</a> Mol Med Rep. 11 (3), 1566-1572 (2015).</li>
<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li>
<li>Li, H. Y., Ren, K., Wang, C., Bu, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin+organoid+research+progress+and+potential+applications%5BTitle%5D)+AND+%22International+Journal+of+Dermatology+and+Venereology%22%5BJournal%5D)">Skin organoid research progress and potential applications.</a> International Journal of Dermatology and Venereology. 5 (2), 101-106 (2022).</li>
<li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+human+mucosal+melanoma+organoid+platform+for+modeling+tumor+heterogeneity+and+exploring+immunotherapy+combination+options%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options.</a> Sci Adv. 9 (43), eadg6686 (2023).</li>
<li>Vlachogiannis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+organoids+model+treatment+response+of+metastatic+gastrointestinal+cancers%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.</a> Science. 359 (6378), 920-926 (2018).</li>
<li>Ganesh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rectal+cancer+organoid+platform+to+study+individual+responses+to+chemoradiation%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">A rectal cancer organoid platform to study individual responses to chemoradiation.</a> Nat Med. 25 (10), 1607-1614 (2019).</li>
<li>Vilgelm, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fine-needle+aspiration-based+patient-derived+cancer+organoids%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Fine-needle aspiration-based patient-derived cancer organoids.</a> iScience. 23 (8), 101408(2020).</li>
</ol>

<table><tbody><tr><td>2 mL Red Cap Internal Threaded Polypropylene Cryogenic Vial</td><td>Corning</td><td>431420</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>100 mm TC-treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>105 mm Polystyrene Forceps Sterile</td><td>Caplugs Evergreen&amp;nbsp;</td><td>222-1121-B1I</td><td></td></tr><tr><td>150 cm&lt;sup&gt;2 &lt;/sup&gt;Cell Culture Flask</td><td>Corning</td><td>CLS430825</td><td></td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Advanced DMEM/F12 (1x) Reduced Serum Medium (1:1)</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>B-27 Supplement (50x)</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Brilliant Violet 510 anti-human Perforin Antibody</td><td>BioLegend</td><td>308120</td><td></td></tr><tr><td>Brilliant Violet 605 anti-human Ki-67 Antibody</td><td>BioLegend</td><td>350522</td><td></td></tr><tr><td>Brilliant Violet 650 anti-human CD366 (Tim-3) Antibody</td><td>BioLegend</td><td>345028</td><td></td></tr><tr><td>Brilliant Violet 711 anti-human CD45 Antibody</td><td>BioLegend</td><td>304050</td><td></td></tr><tr><td>Cell Culture Inserts 0.4 &amp;micro;m, 30 mm Diameter</td><td>Millipore Sigma</td><td>PIHP03050</td><td></td></tr><tr><td>Cell Staining Buffer</td><td>BioLegend</td><td>420201</td><td></td></tr><tr><td>Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning&amp;nbsp;</td><td>352350</td><td></td></tr><tr><td>Collagenase, Type IV, powder</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>Cultrex Rat Collagen I</td><td>Trevigen</td><td>3440-100-01</td><td></td></tr><tr><td>Culture Plate, PS, 48 wells, TC treated with lid, sterile</td><td>Max Scientific&amp;nbsp;</td><td>07-6048</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM - high glucose 4.5 mg/mL</td><td>Corning</td><td>MT10-0130CV</td><td></td></tr><tr><td>Dnase I - Grade II</td><td>Millipore Sigma</td><td>10104159001</td><td></td></tr><tr><td>DPBS, 1%&amp;nbsp;</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine Serum, FBS</td><td>Corning</td><td>MT35-010-CV</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences&amp;nbsp;</td><td>556547</td><td></td></tr><tr><td>Forskolin</td><td>Tocris</td><td>1099</td><td></td></tr><tr><td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td><td>ThermoFisher</td><td>10131035</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>ThermoFisher</td><td>35050061</td><td></td></tr><tr><td>Ham&#039;s F12 Nutrient Mix</td><td>ThermoFisher</td><td>11765054</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Hygromycin B (50 mg/mL)</td><td>ThermoFisher</td><td>10687010</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>L-WRN cell</td><td>ATCC</td><td>CRL-3276</td><td></td></tr><tr><td>Matrigel Phenol Free &amp;amp; Growth Fact. Reduced</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Millex PVDF syringe filter, 0.22 &amp;mu;M</td><td>Millipore Sigma</td><td>SLGVR33RB</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165-5G</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636-100G</td><td></td></tr><tr><td>Nunc TripleFlask Treated Cell Culture Flask</td><td>ThermoFisher</td><td>132867</td><td></td></tr><tr><td>PE/Cyanine5 anti-human CD8a Antibody</td><td>BioLegend</td><td>301010</td><td></td></tr><tr><td>PE-Cy 7 Mouse anti-Human CD279 (PD-1)</td><td>BD Biosciences&amp;nbsp;</td><td>561272</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Human EGF</td><td>Peprotech</td><td>AF-100-15-1MG</td><td></td></tr><tr><td>Recombinant Human FGF-acidic</td><td>Peprotech</td><td>100-17A</td><td></td></tr><tr><td>Sodium Bicarbonate Solution (NaHCO&lt;sub&gt;3&lt;/sub&gt;) (7.5%)</td><td>Quality Biological&amp;nbsp;</td><td>118-085-721</td><td></td></tr><tr><td>Stainless Steel Surgical Blades no. 22</td><td>Integra</td><td>4-322</td><td></td></tr><tr><td>TrypLE Express&amp;nbsp;</td><td>Gibco</td><td>12604-021</td><td></td></tr></tbody></table>

unveiling therapeutic opportunities with melanoma patient-derived organoid models

<meta charset="utf8"></head><body>作者聚焦:使用患者来源的类器官重建黑色素瘤复杂性以进行免疫治疗评估

揭示黑色素瘤患者来源的类器官模型的治疗机会

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

unveiling-therapeutic-opportunities-with-melanoma-patient-derived

Research

JoVE Journal

Cancer Research

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

1.9K 次观看.  University of Pennsylvania. 我们开发和验证黑色素瘤患者来源的类器官。它旨在准确复制肿瘤微环境，研究免疫细胞相互作用，并评估治疗反应，尤其是对免疫疗法的反应。我们正在努力促进对肿瘤生物学的理解，改进药物开发策略，并有可能实现个性化的癌症治疗。准确模拟复杂的肿瘤微环境（包括基质和免疫细胞相互作用）仍然很困难。确保类器官保留原始肿瘤的遗...

视频: 作者聚焦:使用患者来源的类器官重建黑色素瘤复杂性以进行免疫治疗评估

Patient-derived Xenograft Modeling: A Technique to Generate Melanoma Mouse Models

Source: Min Xiao et al. <a href="https://www.jove.com/v/59508"> A Melanoma Patient-Derived Xenograft Model</a>. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted subcutaneously into the flank region of an immunocompromised mouse. The PDX model allows preclinical investigation of melanoma cells that better recapitulate the tumor heterogeneity and melanoma aggressiveness observed in in vivo conditions.

患者来源的异种移植物建模:一种生成黑色素瘤小鼠模型的技术

Source: Min Xiao et al.  A Melanoma Patient-Derived Xenograft Model. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted ...

科研

JoVE Encyclopedia of Experiments

癌症研究

Skin Cancer

Animal model studies

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

3D 器官型黑色素瘤球状皮肤模型

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

黑色素瘤患者来源的异种移植模型

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

为癌症药理学研究创建匹配的体内/体外患者来源的 PDX 和 PDX 衍生类器官模型对

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

为癌症药理学研究创建匹配的体内/体外患者衍生模型对 PDX 和 PDX 衍生器官

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

用于解剖体外肿瘤微环境中免疫反应的微流体共培养模型

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

免疫与感染

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

乳腺上皮细胞球和内皮细胞球体作为乳腺癌研究的潜在功能性体外模型

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

分析 EGFR 突变 NSCLC 患者来源的类器官对靶向治疗的敏感性

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

人源性乳腺类器官的建立和培养

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

用于临床前药物检测的卵巢癌患者来源类器官模型

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

用于临床前药物测试的卵巢癌患者来源的类器官模型

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦:从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

揭示黑色素瘤患者来源的类器官模型的治疗机会

Transcript

Summary

Explore More Videos

患者来源的异种移植物建模:一种生成黑色素瘤小鼠模型的技术

3D 脊髓黑色素瘤球形皮肤模型

黑色素瘤患者衍生异种移植模型

为癌症药理学研究创建匹配的体内/体外患者衍生模型对 PDX 和 PDX 衍生器官

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

患者来源乳腺类器官的建立和培养

用于临床前药物测试的卵巢癌患者来源的类器官模型

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

揭示黑色素瘤患者来源的类器官模型的治疗机会

Transcript

Summary

Explore More Videos

患者来源的异种移植物建模:一种生成黑色素瘤小鼠模型的技术

3D 脊髓黑色素瘤球形皮肤模型

黑色素瘤患者衍生异种移植模型

为癌症药理学研究创建匹配的体内/体外患者衍生模型对 PDX 和 PDX 衍生器官

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

患者来源乳腺类器官的建立和培养

用于临床前药物测试的卵巢癌患者来源的类器官模型

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型