a three-dimensional co-culture model of infiltrating t cells in tumor spheroids

A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

To set up a T cell co-culture, resuspend the appropriate number of T cells in 190 microliters of an appropriate T cell culture medium per well, and tilt the 12-well plate to allow removal of the cell culture medium surrounding the agarose cast first, then in the seeding chamber, without removing the spheroids.
Use a light microscope to check whether any spheroids have been aspirated, and holding the P200 loaded pipette about 1/2 a centimeter above each cast, carefully seed the T cells onto the casts in a dropwise fashion, without dislodging the spheroids. When all of the casts have been seeded, carefully return the plate to the cell culture incubator for 15 minutes, before adding fresh cell culture medium supplemented with fetal bovine serum to the outside of each cast for another 48-hour incubation.

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1. Generation of spheroids
<ol>
	<li>Prepare and autoclave 2% agarose in 1x phosphate-buffered saline (PBS) (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave 35- and 81-microwell rubber molds. 
	CAUTION: Avoid using low-melting agarose for generating the agarose casts for immunohistochemistry (IHC) processing.</li>
	<li>Prepare agarose casts 
	NOTE: 35-microwell casts are used for invasion, immunofluorescence (IF) and cell isolation assays. 81-microwell casts are used for cytotoxicity, IHC, cell isolation and ribonucleic acid (RNA) extraction assays.
	<ol>
		<li>After the agarose has been autoclaved, let molten agarose cool to about 60&#8210;70 &#176;C. In a cell culture hood, use aseptic technique and pipette 500 &#181;L of molten agarose into an 81-microwell rubber mold per well or 330 &#181;L into a 35-microwell rubber mold per well. 
		CAUTION: Avoid creating bubbles while mixing or pipetting agarose. Remove any bubbles by gently pipetting.</li>
		<li>After the agarose is solidified, carefully flex the rubber mold to remove the agarose cast from it. Hereby, place hands above the appropriate well-plate and let the agarose cast drop right into one well of the well-plate. 
		NOTE: Flex the rubber mold at different positions in order to avoid over flexing. It might be helpful to flex the rubber mold and push gently from the bottom at the same time.</li>
		<li>To equilibrate the agarose casts, add cell culture medium, consisting of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (2.5 mL/well for 12-well plate and 1 mL/well for 24-well plate). Put the well-plate into a cell culture incubator (37 &#176;C, 5% CO2) and incubate for 1 h.</li>
	</ol>
	</li>
	<li>Meanwhile, prepare cell culture for seeding. 
	NOTE: The total cell seeding number per agarose cast is to be decided by the investigator. For murine primary pancreatic cancer cell lines, 35,000 cells/35-microwell cast and 81,000 cells/81-microwell cast (in average 1000 cells/spheroids) are seeded for all following assays. The average diameter of a spheroid is about 150&#8210;200 &#181;m after 48 h.</li>
	<li>Remove cell culture medium surrounding the agarose cast with a P1000 pipette first by tilting the well-plate. Then carefully remove the medium in the seeding chamber.</li>
	<li>Carefully seed the prepared tumor cell suspension drop-wise into the cell seeding chamber. Carefully put the well-plate back into the cell culture incubator (37 &#176;C, 5% CO2) for 15 min. 
	NOTE: During this step the cells will settle into the microwells of the agarose cast.</li>
	<li>Add additional medium to the outside of the agarose cast (2.5 mL/well for 12-well plate and 1 mL/well for 24-well plate).</li>
	<li>Put the well-plate back into the cell culture incubator (37 &#176;C, 5% CO2) for 48 h. 
	NOTE: Usually it takes up to a few hours for the cells to form spheroids in the microwells. In general, solid tumor cell spheroids are formed after 24 h and are stable after 48 h. However, this can vary between different cell lines. If necessary, change cell culture medium by carefully tilting the well-plate with the agarose casts and removing the surrounding cell culture medium with a P1000 pipette. Then carefully add fresh medium by pipetting it along the wall of the well. Usually it is not necessary to change the media within the casts due to its small volume and the risk of removing the spheroids.</li>
</ol>
2. Co-culture with T-cells
<ol>
	<li>Resuspend the required number of T-cells in 75 &#181;L (35-microwell cast) or in 190 &#181;L (81-microwell cast) of the appropriate T-cell culture medium (RPMI supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin).</li>
	<li>Remove the cell culture medium surrounding the agarose cast with a P1000 pipette first by tilting the well-plate. Then slowly and carefully remove the medium within the cast by gently targeting one corner of the seeding chamber with a P200 pipette while keeping the well-plate tilted with the other hand.</li>
	<li>(Critical step 1) As there is a risk of removing spheroids, control for loss of spheroids by comparing the number of spheroids within the microwells before and after removing the medium by viewing under the microscope at 10x magnification.</li>
	<li>Carefully seed the T-cell suspension drop-wise into the seeding chamber of the agarose cast by holding the P200 pipette about 0.5 cm above the cast.</li>
	<li>(Critical step 2) There is a risk of flushing out the spheroids while adding the T-cells. Therefore, it is critical to add the T-cells very slowly and about 0.5 cm above the seeding chamber. 
	NOTE: One possibility to decrease the number of flushed out spheroids is by pointing the pipette to one corner of the seeding chamber while adding the T-cells, thus only risking the spheroids in this corner to be flushed out.</li>
	<li>Carefully place the well-plate back into the cell culture incubator (37 &#176;C, 5% CO2) for 15 min.</li>
	<li>Take the well-plate out from the incubator and add fresh cell culture medium (Roswell Park Memorial Institute media (RPMI) supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin) around the agarose cast by slowly pipetting it along the wall of the well.</li>
	<li>Put the well-plate back into the cell culture incubator for 48 h.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Petri Dishes</td>
			<td>Microtissues Inc</td>
			<td>Z764019 &#38; Z764051</td>
			<td>referred to as &#34;rubber molds&#34; in the protocols; 81-microwell &#38; 35-microwell molds</td>
		</tr>
		<tr>
			<td>8-well Chamber Slides</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Type I, low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>11965092, 16000044</td>
			<td>referred to as &#34;cell culture medium&#34; in the protocols</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>ThermoFisher</td>
			<td>11875093</td>
			<td>for T-cell culture medium</td>
		</tr>
	</tbody>
</table>

Source: Lin, Y., et al. <a href="https://app.jove.com/v/61392">Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture.</a> J. Vis. Exp. (2020).
This video demonstrates a method to establish a three-dimensional tumor spheroid and T cell co-culture. An agarose cast with microwells containing tumor spheroids is prepared, followed by the addition of T cells. After incubation, this setup allows studying the interaction of T cells with tumor spheroids in a controlled three-dimensional environment.

1. Generation of spheroids

	Prepare and autoclave 2% agarose in 1x phosphate-buffered saline (PBS) (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave ...

Begin with a culture plate containing an agarose cast. The cast comprises a seeding chamber with multiple microwells.
Each microwell contains a tumor spheroid in a culture medium. The spheroids are three-dimensional aggregates of tumor cells.
First, remove the medium surrounding the agarose cast. Then, remove the medium within the cast from one corner of the seeding chamber to avoid the loss of spheroids.
Introduce a suspension of tumor-specific T cells dropwise into the seeding chamber, allowing the cells to settle into the microwells containing the spheroids.
Next, add a culture medium around the agarose cast and incubate the plate under physiological conditions to develop a co-culture.
During incubation, the T cells surround the spheroid. A subset of T cells infiltrate the spheroid, while others remain in the periphery, establishing a three-dimensional co-culture model.

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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

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癌症研究

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

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Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

免疫与感染

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

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Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

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A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids