The authors thank the staff of the University Hospital animal facility (UTE) of Nantes for animal husbandry and care, the cellular and tissular imaging core facility of Nantes University (MicroPICell) for imaging, and the Cytometry facility (Cytocell) from Nantes for their expert technical assistance. This work was funded by INSERM, CNRS, Universit&#233; de Nantes, Institut National du Cancer (INCa#PLBio2014-155), Ligue Nationale contre le Cancer (AO InterRegional 2017), and the European consortium ERA-Net Transcan2 (Immunoglio). The team is funded by the Fondation pour la Recherche Medicale (DEQ20170839118). This work was realized in the context of the LabEX IGO and the IHU-Cesti programs, supported by the National Research Agency Investissements d'Avenir via the programs ANR-11-LABX-0016-01 and ANR-10-IBHU-005, respectively. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. The authors thank Chirine Rafia for providing help in correcting the manuscript.

The following procedure involving animal subjects was performed according to institutional guidelines (Agreement #00186.02; regional ethics committee of the Pays de la Loire [France]). Human PBMCs were isolated from the blood collected from informed healthy donors (Etablissement Fran&#231;ais du Sang Nantes, France). All steps are performed under sterile conditions.
1. Non-specific Expansion of Cytotoxic Effector T Lymphocytes
<ol>
	<li>Prepare and irradiate feeder cells at 35 Gy. For the st...

<ol>
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An adoptive transfer of selected native or engineered immune effector cells represents a promising approach to efficiently treat tumors, such as infiltrative brain cancers, taking care of limiting reactivities against non-transformed cells15,16,17,18. However, the central nervous system, which comprises the brain, has a particular immune status, notably due to the existence of the blood-brain b...

GBM (WHO grade IV astrocytoma), is the most frequent and aggressive primary brain cancer in adults. In spite of aggressive treatments that combine surgery and radio-chemotherapy, GBM remains associated with an extremely poor prognosis (median survival of 14.6 months and a 2-year-mortality &#62; 73%)1. This evidences that few efficient therapeutic advances have been validated over the last decade2. Among candidates for the design of more effective therapeutic strategies3,4,5, immunotherapies6 are currently explored to track and eliminate highly invasive and radio/chemo-resistant tumor cells, suspected for their key contribution to rapid and fatal tumor relapse7. Various potential immunological targets were identified and proposed for immunotherapies, involving natural or modified &#945;&#946; or &#978;&#948; T lymphocytes such as GBM-specific tumor antigens or stress-induced molecules8,9,10. The possibility to administrate selected GBM-reactive immune cell effectors represents a unique opportunity to deliver elevated amounts of effector lymphocytes directly into the site of residual malignancy. To support this strategy, we have recently shown that models based on immunodeficient mice carrying orthotopic primary human GBM xenografts faithfully recapitulate the development of brain tumors in GBM patients9,11. Moreover, these models were used to demonstrate the strong antitumor efficiency of adoptively transferred allogeneic human V&#978;9V&#948;2T lymphocytes.
This protocol describes the critical experimental steps for achieving stereotactic immunotherapies of brain tumors, such as GBM, based on the adoptive transfer of allogeneic T lymphocytes. The article shows: (i) the amplification of therapeutic allogeneic immune effector T lymphocytes, such as human V&#978;9V&#948;2T lymphocytes; (ii) the preparation of these effector T lymphocytes for injection; (iii) the procedure for stereotactic administration within the brain, near the tumor. This article also provides insight into the behavior of these cellular effectors after stereotactic injection.
The therapeutic approach presented here is based on the injection of 20 x 106 effector cells per dose for each brain tumor-bearing immunodeficient mouse. An initial in vitro expansion step is required to produce large quantities of immune cells. Therefore, non-specific cell expansions are performed using phytohemagglutinin (PHA-L) and irradiated allogeneic feeder cells: peripheral-blood mononuclear cells (PBMCs) from healthy donors and Epstein Barr Virus (EBV)-transformed B-lymphoblastoid cell lines (BLCLs), derived from PBMCs by in vitro infection with EBV-containing culture supernatant from the Marmoset B95-8 cell line, in the presence of 1 &#181;g/mL cyclosporin-A.
GBM-reactive effector immune cells are compared and selected from in vitro assays9. These effector cells are activated and amplified using standard protocols, according to their nature (e.g., human V&#947;9V&#948;2 T lymphocytes9 or human anti-herpes virus &#945;&#946; T lymphocytes12) with a minimum purity of &#62; 80%, as routinely checked by cytometric analysis. The cell expansion procedure detailed below applies to various human lymphocyte subsets.

<table border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBMCs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:232px;">from 3 different healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BLCLs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td>from 3 different donors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Roswell Park Memorial Institute medium (RPMI)</td>
			<td style="width:136px;">Gibco</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">FCS</td>
			<td style="width:136px;">Dutscher</td>
			<td style="width:119px;">S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-glutamine</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">penicillin/streptomycin</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">IL-2</td>
			<td style="width:136px;">Novartis</td>
			<td style="width:119px;">proleukin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PHA-L</td>
			<td style="width:136px;">Sigma</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Stereotaxic frame</td>
			<td>Stoelting Co.</td>
			<td align="right">51600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mouse adaptator for stereotaxic frame&#160;&#160;</td>
			<td>Stoelting Co.</td>
			<td align="right">51624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microsyringe pump injector&#160;</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">UMP3-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil Syringe</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NSG mice</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:119px;">NSGSSFE07S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ketamine</td>
			<td style="width:136px;">Merial</td>
			<td style="width:119px;">Imalg&#232;ne 1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylazine</td>
			<td style="width:136px;">Bayer</td>
			<td style="width:119px;">Rompur 2%</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Scissors</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">201758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Forceps</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">501215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OmniDrill 115/230V</td>
			<td style="width:136px;">WPI</td>
			<td align="right">503598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Vicryl 4-0</td>
			<td style="width:136px;">Ethicon</td>
			<td>VCP397H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylocaine</td>
			<td style="width:136px;">Astrazeneca</td>
			<td>3634461</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

This study describes the strategy of adoptive transfers of cellular immune effector cells within the brain of tumor-carrying mice, based on stereotactic injections performed directly within the tumor bed.
To minimize any risk of brain injury associated with a large injection volume, the effector cell suspension needs to be concentrated (20 x 106 cells in 15 - 20 &#181;L of PBS). To check whether this cell concentratio...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Glioblastoma multiforme remains the most frequent and aggressive primary brain tumor in adults. Despite aggressive treatments based on surgery followed by radio and chemotherapy, GBM is associated with an extremely poor prognosis and a median survival lower than 15 months. Since few therapeutic advances have been made over the last decade, cellular immunotherapies are currently explored to eliminate highly-invasive and resistant GBM cells likely involved in rapid tumor relapse.administrations of selected GBM-reactive cytotoxic immuno factors in the vicinity of the tumor could represent a unique opportunity to deliver concentrated cellular immunotherapy, directly into the site of residual malignancy. Our group recently showed that immunodeficient NSG mice carrying orthotopic primary human GBM xenograft recapitulate GBM to more development in patients. These GBM models were used to evaluate the efficacy of cytotoxic immune effectors on intratumoral injections.This protocol describes our therapeutical process based on first the isolation and amplification of GBM-reactive immune effector cells. Second, the propagation of these effector cells for intercranial injection. And third, their stereotactic injection at the tumor site within the mouse brain.The behavior of the immune effector after orthotopic injection was also investigated. Three weeks after PHA fetus stimulation, effector cells, which form cell clump in the bottom of the flask should have returned to rest state and are ready to be used. After checking effector cell count, resuspend and collect cells in 115 milliliters tube and centrifuge them.Carefully and completely remove a supernatant. And resuspend cells in sterile PBS and centrifuge to wash them. Carefully and completely take off the supernatant.And then resuspend cells in one milliliter of sterile PBS. Transfer cells in a microtube for centrifugation. Again, carefully and completely take off the supernatant by pipetting slowly.The next step is critical, resuspend cells in half of the final volume of sterile PBS and homogenize softly and carefully. Verify the final volume with micropipette, final volume per mouse has to be between 15 and 20 microliter for 20 million cells. Be sure it will not exceed 20 microliter per mouse.Keep cells on ice until injection. Ensure that the animal is adequately anesthetized by a toe pinch and then remove fur from the surgical site, between the two ears and up to the nose. Resuspend cells slowly to prevent cell clumping and load cell suspension into syringe with great care to avoid the presence of bubbles.Then, place the syringe into the adapted syringe pump. Disinfect the surgical site with salt and povidone iodine 5%solution and place a lubricating ophthalmic ointment in the mouse's eyes to prevent drying of the cornea. Then, position the anesthetized mouse on the stereotactic frame on a warm block.Appropriately position mouse's nose and teeth above the tooth bar. Tighten ear bars firmly in the mouse ears to immobilize the head. Be careful not to damage the eardrums, compromise the respiration, and make sure that head is well immobilized.Make a midline sagittal skin incision with sterile scissors along the upper part of the cranium to expose the skull. Identify the intersection of the sagittal and coronal structures to serve as landmarks for stereotactic localization, and place the syringe above this point. Move the syringe at predetermined coordinates.Two millimeter right lateral, and 0.5 millimeter anterior of the Using a microdrill, make a small hole in the skull with sterile drill bit. Be careful to remain superficial in order to avoid brain injury. Insert syringe carefully into the drilled hole.Move slowly forward the needle three millimeter down in the dura, and then backward 0.5 millimeter to a final depth of 2.5 millimeter cells injection. Run the cell injection at two or three microliter per minute, and monitor the mice all along the injection time. Once injection is complete, withdraw the needle for only one millimeter and keep syringe in place for an additional minute before slowly withdrawing completely the syringe to prevent any leakage from the infusion site.Remove carefully the animal from the stereotactic frame. Close skin with appropriate surgical suture and apply 2%Lidocaine gel directly on the wound. Transfer anesthetized mouse to its respective cage above heating pad set to 37 degrees Celsius to maintain appropriate mouse body temperature until full recovery from anesthesia.As the suspension is very concentrated, the cell viability and reactivity were checked. Effector cells were loaded in syringe in order to mimic in vivo injection. Cells were collected immediately or 10 minutes later and were analyzed by flow cytometry for propidium iodide staining.As you can see, the preparation and the loading did not affect the viability of effector cells at 24 hours. The reactivity against Glioblastoma tumor cells were also checked using either unprepared cells or effectors prepared and stayed three hours on the eyes. Reactivity is monitored by positive staining of the activation marker CD107A.These results demonstrate that the effector cells'reactivity were not affected by the preparation. Seven days after effector cells'injection, mice brains were collected, sectioned, and stained for hematic coloration to allow the identification of tumor structure. Then anti-human CD3 staining allowed localization of effector cells.Interestingly, effector cells were found around the tumor and in the tumor core, but also in contralateral hemisphere. Human effector T-cells isolated from mouse brain 48 hours after the injection represent 2%of brain cells. They were still able to proliferate in presence of PHA, feeders and IL2 stimulation, with a 99%purity after amplification.Thanks to this protocol, resting human effector cells can survive and patrol with a mouse brain parenchyma for several days following the stereotaxic injection. In conclusion, adoptive transfer of human effector cells by stereotactic injections represent promising approaches to efficiently treat infiltrative malignant brain tumors with limited deleterious effects on healthy cells. This route of administration takes advantage that human cells are delivered closely to the tumor site, limiting their dilution within the tissues and the organism.Here we described the procedure following the delivering of a large amount of immune cells within the brain tumor without harmful effects on effector cells and, importantly, without any visible deleterious effect on mice. Moreover, locally-injected immune cells in mouse brain are not only able to survive and keep their ability to be activated and amplified ex vivo, but also to and eliminate disseminated tumor cells in the surrounding brain tissue. Collectively, these features are essential to allow the elimination of strong infiltrative tumor cells that characterize GBM tumors.This protocol opens new opportunity for the establishment of adoptive transfer procedure in mice model of brain tumor. Essential step before considering clinical CD.

stereotactic-adoptive-transfer-cytotoxic-immune-cells-murine-models

Research

JoVE Journal

Immunology and Infection

7.7K Views.  CRCINA. Glioblastoma multiforme remains the most frequent and aggressive primary brain tumor in adults. Despite aggressive treatments based on surgery followed by radio and chemotherapy, GBM is associated with an extremely poor prognosis and a median survival lower than 15 months. Since few therapeutic advances have been made over the last decade, cellular immunotherapies are currently explored to eliminate highly-invasive and resistant GBM cells likely involved in rapid tumor relapse.administ...