Name: paramyxoviruses for tumor-targeted immunomodulation: design and evaluation ex vivo
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo at JoVE.com

These methods were established in the Virotherapy Group led by Prof. Dr.&#160;Dr. Guy Ungerechts at the National Center for Tumor Diseases in Heidelberg. We are indebted to him and all members of the laboratory team, especially Dr. Tobias Speck, Dr. R&#363;ta Veinalde, Judith F&#246;rster, Birgit Hoyler, and Jessica Albert. This work was supported by the Else Kr&#246;ner-Fresenius-Stiftung (Grant 2015_A78 to C.E. Engeland) and the German National Science Foundation (DFG, grant EN 1119/2-1 to C.E. Engeland). J.P.W. Heidbuechel receives a stipend by the Helmholtz International Graduate School for Cancer Research.

NOTE: [O], [P], and [M] indicate subsections applicable to: OVs in general, (most) paramyxoviruses, or MV only, respectively. [B] indicates sections specific for BTE transgenes.
1 Cloning of Immunomodulator-encoding Transgenes into Measles Virus Vectors
<ol>
	<li>[O] Design insert sequence.
	<ol>
		<li>[O] Decide on an immunomodulator of interest based on literature research or on exploratory data such as genetic screens41 and derive ...

Oncolytic immunotherapy (i.e., OVT in combination with immunomodulation) holds great promise for cancer treatment, demanding further development and optimization of oncolytic viruses encoding immunomodulatory proteins. This protocol describes methods to generate and validate such vectors for subsequent testing in relevant preclinical models and potential future clinical translation into novel anti-cancer therapeutics.
Numerous different oncolytic virus platforms with distinct advantag...

Oncolytic viruses (OVs) are being developed as anti-cancer therapeutics that specifically replicate within and kill tumor cells while leaving healthy tissues intact. It has now become common understanding that oncolytic virotherapy (OVT), in most cases, does not rely solely on complete tumor lysis by efficient replication and spreading of the virus, but requires additional mechanisms of action for treatment success, including vascular and stromal targeting and, importantly, immune stimulation1,2,3,4. While many early OV studies used&#160;unmodified viruses, current research has profited from an improved biological understanding, virus biobanks that potentially contain novel OVs, and the possibilities offered by genetic engineering in order to create advanced OV platforms5,6,7.
Given the recent success of immunotherapy, immunomodulatory transgenes are of particular interest regarding the genetic engineering of OVs. Targeted expression of such gene products by OV-infected tumor cells reduces toxicity compared to systemic administration. Targeting is achieved either by using viruses with inherent oncoselectivity or by modifying viral tropism8. Local immunomodulation enhances the multi-faceted anti-tumor mechanisms of OVT. Furthermore, this strategy is instrumental in interrogating the interplay between viruses, tumor cells, and the host immune system. To this end, this protocol provides an applicable and adjustable workflow to design, clone, rescue, propagate, and validate oncolytic paramyxovirus (specifically measles virus) vectors encoding such transgenes.
Modulation of the immune response can be achieved by a wide variety of transgene products targeting different steps of the cancer-immunity cycle9, including enhancing tumor antigen recognition [e.g., tumor-associated antigens (TAAs) or inducers of major histocompatibility complex (MHC) class I molecules] over supporting dendritic cell maturation for efficient antigen presentation (cytokines); recruiting and activating desired immune cells such as cytotoxic and helper T cells [chemokines, bispecific T cell engagers (BTEs)]; targeting suppressive cells such as regulatory T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and cancer-associated fibroblasts (antibodies, BTEs, cytokines); and preventing effector cell inhibition and exhaustion (checkpoint inhibitors). Thus, a plethora of biological agents is available. Evaluation of such virus-encoded immunomodulators regarding therapeutic efficacy and possible synergies as well as understanding of respective mechanisms is necessary to improve&#160;cancer therapy.
Negative sense single-stranded RNA viruses of the Paramyxoviridae family are characterized by several features conducive to their use as oncolytic vectors. These include a natural oncotropism, large genomic capacity for transgenes (more than 5 kb)10,11, efficient spreading including syncytia formation, and high immunogenicity12. Therefore, OV platforms based on canine distemper virus13, mumps virus14, Newcastle disease virus15, Sendai virus16,17, simian virus 518, and Tupaia paramyxovirus19 have been developed. Most prominently, live attenuated measles virus vaccine strains (MV) have progressed in preclinical and clinical development20,21. These virus strains have been used for decades for routine immunization with an excellent safety record22. Moreover, there is no risk for insertional mutagenesis due to the strictly cytosolic replication of paramyxoviruses. A versatile reverse genetics system based on anti-genomic cDNA which allows for insertion of transgenes into additional transcription units (ATUs) is available11,23,24. MV vectors encoding sodium-iodide symporter (MV-NIS) for imaging and radiotherapy or soluble carcinoembryonic antigen (MV-CEA) as a surrogate marker for viral gene expression are currently being evaluated in clinical trials (NCT02962167, NCT02068794, NCT02192775, NCT01846091, NCT02364713, NCT00450814, NCT02700230, NCT03456908, NCT00408590, and NCT00408590). Safe administration has been confirmed and cases of anti-tumor efficacy have been reported in previous studies25,26,27,28,29,30 (reviewed by Msaouel et al.31), paving the way for additional oncolytic measles viruses that have been developed and tested preclinically. MV encoding immunomodulatory molecules targeting diverse steps of the cancer-immunity cycle have been shown to delay tumor growth and/or prolong survival in mice, with evidence for immune-mediated efficacy and long-term protective immune memory in syngeneic mouse models. Vector-encoded transgenes include granulocyte-macrophage colony stimulating factor (GM-CSF)32,33, H. pylori neutrophil-activating protein34, immune checkpoint inhibitors35, interleukin-12 (IL-12)36, TAAs37, and BTEs38, which cross-link a tumor surface antigen with CD3 and thus induce anti-tumor activity by polyclonal T cells, irrespective of T cell receptor specificity and co-stimulation (Figure 1). The promising preclinical results obtained for these constructs demand further translational efforts.
Talimogene laherparepvec (T-VEC), a type I herpes simplex virus encoding GM-CSF, is the only oncolytic therapeutic approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA). The phase III study leading to approvals in late 2015 has not only shown efficacy at the site of intra-tumoral injection, but also&#160;abscopal effects (i.e., remissions of non-injected lesions) in advanced melanoma39. T-VEC has since entered additional trials for application in other tumor entities (e.g., non-melanoma skin cancer, NCT03458117; pancreatic cancer, NCT03086642) and for evaluation of combination therapies, especially with immune checkpoint inhibitors (NCT02978625, NCT03256344, NCT02509507, NCT02263508, NCT02965716, NCT02626000, NCT03069378, NCT01740297, and Ribas et al.40).
This demonstrates not only the potential of oncolytic immunotherapy but also the need for further research to identify superior combinations of OVT and immunomodulation. Rational design of additional vectors and their development for preclinical testing is key to this undertaking. This will also advance understanding of underlying mechanisms and has implications for the progression towards more personalized cancer treatment. To this end, this publication presents the methodology for the modification and development of paramyxoviruses for targeted cancer immunotherapy and, more specifically, of oncolytic measles viruses encoding T cell-engaging antibodies (Figure 2).

<table border="0" cellpadding="0" cellspacing="0" style="width:833px;" width="833">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Rapid DNA Dephos &#38; Ligation Kit</td>
			<td style="width:312px;">Roche Life Science, Mannheim, Germany</td>
			<td style="width:123px;">4898117001</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CloneJET PCR Cloning Kit</td>
			<td style="width:312px;">Thermo Fisher Scientific, St. Leon-Rot</td>
			<td style="width:123px;">K1231</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Agarose</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">A9539-500G</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Gel Extraction Kit</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">28704</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">NEB 10-beta Competent E. coli</td>
			<td style="width:312px;">New England Biolabs (NEB), Frankfurt/Main, Germany</td>
			<td style="width:123px;">C3019I</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">LB medium after Lennox</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">X964.1</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ampicillin</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">HP62.1</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Miniprep Kit</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">27104</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Restriction enzyme HindIII-HF</td>
			<td style="width:312px;">New England Biolabs (NEB), Frankfurt/Main, Germany</td>
			<td style="width:123px;">R3104S</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:312px;">Invitrogen, Darmstadt, Germany</td>
			<td style="width:123px;">31966-021</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fetal bovine serum (FBS)</td>
			<td style="width:312px;">Biosera, Boussens, France</td>
			<td style="width:123px;">FB-1280/500</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:244px;">FugeneHD</td>
			<td style="width:312px;">Promega, Mannheim, Germany</td>
			<td style="width:123px;">E2311</td>
			<td style="width:155px;">may be replaced by transfection reagent of choice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Kanamycin</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">K0129</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Vero cells</td>
			<td style="width:312px;">ATCC, Manassas, VA, USA</td>
			<td style="width:123px;">CCL81</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">B16-CD46/ B16-CD20-CD46</td>
			<td style="width:312px;">J. Heidbuechel, DKFZ Heidelberg</td>
			<td style="width:123px;"></td>
			<td style="width:155px;">available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Granta-519</td>
			<td style="width:312px;">DSMZ, Braunschweig, Germany</td>
			<td style="width:123px;">ACC 342</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Opti-MEM (serum-free medium)</td>
			<td style="width:312px;">Gibco Life Technologies, Darmstadt, Germany</td>
			<td style="width:123px;">31985070</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Colorimetric Cell Viability Kit III (XTT)</td>
			<td style="width:312px;">PromoKine, Heidelberg, Germany</td>
			<td style="width:123px;">PK-CA20-300-1000</td>
			<td style="width:155px;">includes XTT reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dulbecco&#39;s Phosphate-Buffered Saline (PBS)</td>
			<td style="width:312px;">Gibco Life Technologies, Darmstadt, Germany</td>
			<td style="width:123px;">14190-094</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">QIAquick Ni-NTA Spin Columns</td>
			<td style="width:312px;">QIAGEN, Hilden, Germany</td>
			<td style="width:123px;">31014</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium chloride</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">3957.3</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Imidazole</td>
			<td style="width:312px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:123px;">I5513-25G</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Amicon Ultra-15, PLGC Ultracel-PL Membran, 10&#160;kDa</td>
			<td style="width:312px;">Merck, Darmstadt, Germany</td>
			<td style="width:123px;">UFC901024</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">BCA Protein Assay Kit</td>
			<td style="width:312px;">Merck Milipore</td>
			<td style="width:123px;">71285-3</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">IgG from human serum</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">I4506</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-HA-PE</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-092-257</td>
			<td style="width:155px;">RRID: AB_871939</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:244px;">Mouse IgG1, kappa Isotype Control, Phycoerythrin Conjugated, Clone MOPC-21 antibody</td>
			<td style="width:312px;">BD Biosciences, Heidelberg, Germany</td>
			<td style="width:123px;">555749</td>
			<td style="width:155px;">RRID: AB_396091</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-HA-biotin antibody, clone 3F10</td>
			<td style="width:312px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:123px;">12158167001</td>
			<td style="width:155px;">RRID: AB_390915</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Anti-Biotin MicroBeads</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-090-485</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MS Columns</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-201</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MiniMACS Separator</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-102</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">MACS MultiStand</td>
			<td style="width:312px;">Miltenyi Biotech, Bergisch Gladbach, Germany</td>
			<td style="width:123px;">130-042-303</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">RIPA buffer</td>
			<td style="width:312px;">Rockland Immunochemicals, Gilbertsville, PA, USA</td>
			<td style="width:123px;">MB-030-0050</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:244px;">CytoTox 96 Non-Radioactive Cytotoxicity Assay</td>
			<td style="width:312px;">Promega, Mannheim, Germany</td>
			<td style="width:123px;">G1780</td>
			<td style="width:155px;">includes 10x lysis solution, substrate solution (substrate mix and assay buffer), and stop solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cell lifter</td>
			<td style="width:312px;">Corning, Reynosa, Mexico</td>
			<td style="width:123px;">3008</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10 cm dishes</td>
			<td style="width:312px;">Corning, Oneonta, NY, USA</td>
			<td style="width:123px;">430167</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">15 cm dishes</td>
			<td style="width:312px;">Greiner Bio-One, Frickenhausen, Germany</td>
			<td style="width:123px;">639160</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">96-well plates, U-bottom</td>
			<td style="width:312px;">TPP, Trasadingen, Switzerland</td>
			<td style="width:123px;">92097</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">96-well plates, flat bottom</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353072</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">6-well plates</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353046</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">12-well plates</td>
			<td style="width:312px;">Neolab, Heidelberg, Germany</td>
			<td style="width:123px;">353043</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">50 mL tubes</td>
			<td style="width:312px;">nerbe plus, Winsen/Luhe, Germany</td>
			<td style="width:123px;">02-572-3001</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">T175 cell culture flasks</td>
			<td style="width:312px;">Thermo Fisher Scientific, St. Leon-Rot</td>
			<td style="width:123px;">159910</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">0.22 &#181;m filters</td>
			<td style="width:312px;">Merck, Darmstadt, Germany</td>
			<td style="width:123px;">SLGPM33RS</td>
			<td style="width:155px;"></td>
		</tr>
	</tbody>
</table>

paramyxoviruses for tumor-targeted immunomodulation: design and evaluation ex vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

Figure 1&#160;illustrates the mechanism of action of oncolytic measles viruses encoding bispecific T cell engagers. A flowchart depicting the workflow of this protocol is presented in Figure 2. Figure 3 shows an example of a modified oncolytic measles virus genome. This provides a visual representation of the specific changes applied to the measles virus anti-genome and particular fe...

Watch this Scientific Journal Video about Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo at JoVE.com

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

This method can help address key challenges in the field of cancer immunotherapy by facilitating the development of oncolyctic vectors for targeted immunomodulation. The main advantage of the applied reverse genetic system is its versatility;therefore, vectors can be adapted to address various research questions and target tumors with diverse immune signatures. The virus propagation steps especially determining the optimal time point to harvest virus are difficult to learn from a text protocol because syncytia formation must be assessed visually over time.To begin design and clone recombinant immunomodulatory vectors as described in the manuscript. This protocol describes specific steps for the development of an oncolytic measles vaccine vector, encoding a bispecific T cell engager. To rescue measles virus from cDNA, 24 hours before transfection plate measles virus producer cells evenly on a 6 wall plate.Seed 200, 000 cells in 2 milliliters DMEM containing 10 percent FBS per well to achieve 65 to 75 percent confluency at the time of transfection. Mix 5 micrograms of recombinant DNA encoding the measles virus antigenome that will be used to transfect the cells. Appropriate plasmids and a fluorescent reporter in a total volume of 200 microliters of DMEM.Add 18.6 microliters of liposomal transfection reagent to the mixture and immediately flick the tube to mix. Incubate this transfection mix for 25 minutes at room temperature. To transfect the MV producer cells remove the medium from the 6 well plate grown to 65 to 75 percent confluency and add 1.8 milliliters of DMEM with 2 percent FBS and 50 micrograms per milliliter Kanamycin per well.Then add the transfection mix dropwise to each well and swirl carefully. Incubate the cells overnight at 37 degrees Celsius and 5 percent carbon dioxide. On the following day replace the medium with 2 milliliters of fresh DMEM with 2 percent FBS and 50 micrograms per milliliter Kanamycin and repeat this when medium becomes acidic recognized by the yellow color.Use a microscope to observe cells daily for reporter gene expression and syncytia formation. When large syncytia consisting of 20 or more cells are visible or when cells become too dense harvest the virus. 24 hours before the anticipated harvest seed approximately 1.5 million MV producer cells in 12 milliliters of DMEM with 10 percent FBS on 10 centimeter dishes to achieve 65 to 75 percent confluency at the time of virus inoculation.To collect the virus progyny use a cell scraper to carefully scrape adherent producer cells from the 6 wall plate with transfected cells. Transfer the medium containing the scraped cells into a centrifuge tube. Centrifuge for at least 2500 times G and 4 degrees Celsius for 5 minutes to remove cell debris.After centrifugation mix the cell-free supernatant with serum free medium to a final volume of 4 milliliters to prepare inoculum. Remove the medium from the 10 centimeter dish with MV producer cells and add the inoculum to the cells. Incubate for at least 2 hours at 37 degrees Celsius and 5 percent carbon dioxide.After incubation add 6 milliliters of DMEM with 10 percent FBS and incubate cells overnight. Then replace the medium with 12 milliliters of fresh DMEM with 10 percent FBS and incubate at 37 degrees Celsius and 5 percent carbon dioxide until harvesting. Observe the cells at least twice daily.Before syncytia burst when membrane disruption become visible harvest the virus. To harvest the first passage remove supernatant from the plate and add 600 microliters of serum free medium. Then use a cell lifter to scrape the cells and transfer to a clean tube.Immediately after that freeze the virus suspension in liquid nitrogen and store at 80 degrees Celsius for at least 24 hours to ensure thorough freezing. To determine titers of virus stocks in octuplicates per aliquot using 96 wall plates. First pull the producer cells from T75 cell culture flasks into 50 milliliter conical tube.Count the cells and adjust the cell suspension to 150, 000 cells per milliliter in DMEM with 10 percent FBS. Add 90 microliters of DMEM with 10 percent FBS per well of a 96 wall plate. Then add 10 microliters from one aliquot of the virus stock to all 8 wells in the first column of the plate.Mix thoroughly by pipetting up and down at least 10 times. Use a multichannel pipette to transfer 10 microliters of each well, from the first to the second column, and mix thoroughly by pipetting up and down. Repeat this for each column to obtain serial tenfold dilutions of the virus using fresh pipette tips for each dilution step.Discard 10 microliters from each well of the last column. Add 100 microliters of cell suspension with 150, 000 cells per milliliter to each well making sure there are no cell clumps. Incubate at 37 degrees Celsius, 5 percent carbon dioxide for 48 hours.Check for syncytia using a light microscope. Count syncytia in each well of the column with the highest dilution factor containing visible syncytia. For viruses encoding a fluorescent reporter, syncytia can be counted using fluorescence microscopy.For analysis of measles virus replication kinetics one day prior to infection seed 100, 000 vero cells in 1 milliliter of DMEM with 10 percent FBS per well on 12 wall plates with at least 2 wells for each time point of interest. To infect the cells replace the medium with 300 microliters of serum free medium containing the respective amount of the virus. Incubate at 37 degrees Celsius and 5 percent carbon dioxide for at least 2 hours.Remove inoculum and add 1 milliliter of DMEM with 10 percent FBS and continue incubation. At relevant time points after infection harvest viral progyny by directly scraping cells into the medium. Transfer contents from each well to an individual tube.Snap freeze in liquid nitrogen and store at 80 degrees Celsius until titration and then proceed as described in the manuscript. To begin evaluation of BTE induced cell mediated cytotoxicity by LDH release, first isolate secreted transgene products expressed by virus infected target cells and isolate immune effector cells as described in the manuscript. Then seed target cells in a U-bottom 96 wall plate and include samples as described in the manuscript.Add previously isolated immunomodulators at desired concentrations to the respective samples. Add immune defector cells at desired ratios and add medium to a total volume of 100 microliters per well. Incubate for the desired previously determined time frames at 37 degrees Celsius and 5 percent carbon dioxide.45 minutes before sample collection add 10 microliters of 10X Lysis Solution to wells containing T max samples and the corresponding medium controls and continue incubation. Centrifuge the cells at 250 times G for 4 minutes. Transfer 50 microliters of supernatant from each well to wells of a flat bottom 96 wall plate making sure not to transfer the cells.After preparing substrate solution according the manufacturer add 50 microliters to each well. Incubate at room temperature in the dark for 30 minutes or until T max samples turn deep red. After incubation add 50 microliters of stop solution to each well.Centrifuge for 1 minute at 4000 times G and then use a hollowed needle to remove air bubbles. Measure optical absorbance at 490 nanometers and calculate as described in the manuscript. After inoculation with unmodified and BTE encoding oncolytic measles viruses growth curves of the compared vectors on vero cells appear similar.Cell viability curves on mc38 murine colorectal carcinoma cells stably expressing human carcinoembryonic antigen and the MV receptors CD46 after inoculation show that lytic activity of the transgene encoding virus lags behind in the murine tumor cell line. Flow cytometry of target antigen expressing cells incubated with BTE's at five different dilutions showed BTE binding by cells in a concentration dependent manner. In immunoblot after magnetic pull down of BTE associated cells showed that when non targeting BTE's were used there were no detectable amounts of cells;whereas when targeting BTE samples were used bound cells were present in the elution fraction.BTE mediated target antigen specific and concentration dependent cytotoxicity of murine T cells is indicated by representative LDH release assay and shows that in the present example 15 percent specific cell killing was achieved at a relatively high BTE concentration of 1 microgram per milliliter compared to reference cells. While attempting this procedure, it is important to remember to regularly monitor cells to check progression of infection. Don't forget that recombinant measles vaccine strains though attenuated may be hazardous especially to immuno-suppressed individuals.Avoid exposure by following the appropriate bio safety procedures. Individuals handling viruses should be vaccinated before performing these methods. Following this procedure viral expression of further transgenes can be achieved in order to analyze virus hosts interactions and therapeutic effects.The development of these techniques has paved the way for researchers in the field of biotherapy to explore the effects of locally expressed immunomodulators in numerous tumorous entities;In Vitro, In Vivo, and also in clinical trials.

paramyxoviruses-for-tumor-targeted-immunomodulation-design-evaluation

Research

JoVE Journal

Cancer Research

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&amp;#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...