This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.
CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3 as well as resident T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.
CD8 T cells play a key role in the battle against cancer. However, many suppressive mechanisms prevent T lymphocytes from killing malignant cells. Over the last years, several promising strategies that release the brakes on T cells have been developed and used in the clinic5. The two approaches that retain considerable attention are immune checkpoint-targeting antibodies, such as anti-PD-1 and adoptive T cell therapies. Nevertheless, despite recent successes, only a subset of patients benefit from these therapies6. A major challenge is to identify mechanisms of resistance to cancer immunotherapy in order to develop more efficient treatments. Another challenge is to develop model systems in which novel therapies can be characterized and tested for their potency and safety. So far, most of these assays are based on in vitro cell culture systems that poorly mimic the complexity of the tumor tissue.
Ex vivo tissue slices is a promising technique, as it preserves the original tissue microenvironment7. Over the years, we have set up this approach to track the dynamics of T lymphocytes in various tissues. Our results indicate that fluorescently labeled T cells added atop murine lymph node slices migrate into the tissue and respond to their appropriate microenvironmental cues1,2. Similarly, T lymphocytes overlaid onto human lung tumor slices are rapidly recruited into the tumor stroma3. Using this technique, we have identified the extracellular matrix as an important element in controlling the distribution and migration of T cells within human tumors3,4. Because the behavior of ex vivo purified and plated T cells does not necessarily reflect the behavior of resident intratumoral T lymphocytes, we have recently refined this approach4.
Here we describe a protocol to track resident T lymphocytes within slices made from fresh human lung tumors. The different steps consist of the preparation of human lung tumor slices (including agarose embedding and vibratome sectioning of the embedded tissue), the staining of endogenous T cells with fluorescent antibodies in fresh tumor slices, and the monitoring of these cells with a confocal microscope.
Human tumors were obtained with the agreement of the French ethical committee and by the Assistance Publique-Hôpitaux de Paris (AP-HP) in application with the article L.1121-1 of the French law. A written informed consent was obtained from the patients prior to inclusion in the study.
1. Obtaining human lung tumors
2. Agarose embedding and vibratome slicing of human lung tumors
NOTE: Perform all steps until 2.10 under sterile conditions.
3. Immunostaining of resident CD8 T cells and tumor cells
4. Imaging and analyzing resident CD8 T cell migration within human lung tumor slices
NOTE: The confocal microscope used in this protocol is an upright spinning disk equipped with a 25x water immersion objective (25x/0.95 N.A.).
Human lung tumors are usually highly infiltrated in CD8 T cells that are preferentially found in the stroma3,4. By following this protocol one should expect to visualize a large number of immunostained CD8 T in human lung tumor slices. A co-labeling with anti-EpCAM and anti-CD90 antibodies should permit to delineate tumor islets and stromal areas. Fibrillar collagen, abundant in the tumor stroma, can be visualized without exogenous staining by using second harmonic generation on a two-photon microscope3,4. Note that some human lung tumors present no clear distinction between tumor islets and stroma. Such tumors should be discarded as well as specimen presenting large areas of cell necrosis identified by non-specific binding of antibodies and DAPI staining. Consistent with published results obtained in human lung and ovarian carcinomas, resident CD8 T cells are more concentrated in the stroma compared to tumor islets4. A representative example of CD8 T cell distribution in relation to tumor cells and CD90 is shown in Figure 2.
The slice assay allows for the analysis of resident T cell motility in different tumor regions. Useful criteria for comparing CD8 T cell motility in different regions include the average velocity (path length divided by the time duration sampled) and the displacement length (the vector between the beginning and end of a track). Although scarce in tumor islets, CD8 T cells migrate more actively in this compartment compared to the stroma (Figure 3). On average, the mean velocity of individual CD8 T cells should be close to 4 µm/min in the tumor stroma with high intra and inter-tumor variabilities4. In the tumor stroma, the motility of resident T cells is strongly influenced by the density and orientation of extracellular matrix fibers3,4. A successful experiment will result in less T cells present in dense matrix areas compared to loose matrix regions, consistent with published results3,4.
Note that some human lung tumors exhibit a very high level of autofluorescence precluding imaging of resident T cells. Such feature is rapidly observed at the beginning of the imaging session. Autofluorescent tumors should be discarded. A representative example of resident CD8 T cells in an autofluorescent tumor is shown in Figure 4.
Figure 1: Photographs of the perfusion system. A) The phenol red-free RPMI solution is enriched in 5% C02, 95% O2. B) The solution is then perfused by a peristaltic pump. C) The RPMI solution enters the culture dish via the inlet. At the outlet, the solution is aspirated by the pump from the chamber into a waste container. Note that the aspiration tip is set at a level to determine solution height. Please click here to view a larger version of this figure.
Figure 2: Distribution of resident CD8 T cells in a human lung tumor. Representative image of a human lung tumor slice stained for CD8 (green), EpCAM (blue) and CD90 (red). Please click here to view a larger version of this figure.
Figure 3: Motility of resident CD8 T cells in a human lung tumor. Trajectories of individual resident CD8 T cells in the stromal (red) and tumor cell (blue) regions of a human lung carcinoma slice. The slice was stained with the indicated antibodies and imaged for 20 min with a confocal microscope. The fibrillary collagen was detected at the end by using second harmonic generation (SHG) on a two-photon microscope. Tracks are color-coded according to CD8 T cell displacement length. This image has been modified from 4. Please click here to view a larger version of this figure.
Figure 4: Representative image of an autofluorescent human lung tumor. The tumor slice was stained for CD8 (green) and EpCAM (blue). The SHG signal is in red. Please click here to view a larger version of this figure.
A straightforward, quick, and robust technique for generating human lung tumor slices and assessment of the dynamic behavior of resident CD8 T cells in a preserved tumor environment using a confocal microscope is described. This protocol is a refinement of a previous JoVE article that describes the monitoring of plated T cells onto murine lymph node slices2.
Bleaching of fluorescent dyes must be considered especially with first generation dyes like fluorescein isothiocyanate and phycoerythrin. We found that a pronounced photobleaching of directly-coupled antibodies occurred with a two-photon microscope. Conversely, minimal photobleaching was noticed with confocal microscopes, especially using bright fluorescent dyes developed recently.
Antibodies are relatively big molecules and therefore their penetration into the tissue can be difficult. This step can be improved by increasing the incubation time. In addition, the use a smaller staining reagents such as directly-coupled Fab fragments of antibodies represents a good alternative.
All antibodies described in this protocol have been previously validated and known to produce bright staining4. Therefore, isotype controls are not required. However, if modifications to this method using other antibodies are desired, then the use of isotype control antibodies is highly recommended.
This experimental system presents some limitations. The cutting procedure will damage the tissue, which may affect the behavior of CD8 T cells, especially near the cut surface. In addition, a number of soluble factors (e.g. chemokines) instrumental in controlling the migration of T cells can be lost. One way to bypass these problems is by monitoring T cells located at several tens of microns from the surface in presumably healthier regions of the tissue. Published experiments reveal that T cells localized deep within the tissue migrate more actively than T cells localized near the cut surface4. We have used confocal microscopes for our studies but it is likely that two-photon microscopes will increase the spatial resolution in depth.
This approach relies on the use of fluorescently-coupled antibodies that are not neutral molecules. Full-sized antibodies are susceptible to affect the normal functioning of T cells in different ways. First, anti-CD8 antibodies can alter T cell migration by triggering an intracellular signaling cascade capable of affecting the cytoskeleton of the lymphocyte. Second, anti-CD8 antibodies can modulate the interaction between CD8 and MHC class I molecules, an important step in the antigen recognition process. Third, intact antibodies can bind to Fc receptors expressed by many myeloid cells distributed in the tumor and therefore susceptible to increase the non-specific signal. We have no indication that such a problem affected our data, probably because Fc receptors of resident myeloid cells are, unlike cells in culture, saturated with immunoglobulins present within the tumor milieu. Indeed, the addition of serum during the labeling procedure, a process known to block free Fc receptors, did not change the immuno-staining. Nonetheless, Fab fragments of antibodies, devoid of Fc region, as well as antibodies mutated in their Fc region and camelid-derived antibody fragments8 represent other alternative strategies to track resident cells with fluorescent tracers.
Over the last decades, several model systems have been developed and used for the real-time imaging of T lymphocytes. These include in vitro preparations of T cells previously purified and cultured with antigen-presenting cells. Such preparations have allowed remarkable progress in defining the mechanisms of antigen recognition and responsiveness. However, they lack the complexity of the tissue environment. Other preparations have been established that more closely mimic the structure of a native tissue. For examples, three-dimensional collagen gel matrices in which purified T cells have been embedded, as well as culture of reaggregated tissue fragments have been used to monitor the dynamic behavior of T lymphocytes with fluorescent imaging technologies9. These systems present the advantage of an architecture close to the native tissue, but they lack other important cellular and soluble factors. In mouse, two-photon intravital microscopy has been employed to track T cells in the truly physiological environment of an intact organ10. However, such approach is technically difficult to set up. In addition, mouse models show notable differences with the human physiology.
The technique described in this protocol represents a complex multi-cellular tumor environment that is uniquely positioned to act as an important link between co-culture studies and animal models.
The protocol described herein can also be used for tracking the motility of other cells than CD8 T lymphocytes for which antibodies have been generated. Furthermore, the technique can be adapted to other human and murine tumors and tissues. For instance, we have been able to image in real time the dynamics of B cells labeled with an anti-CD19 antibody in fresh human tonsils.
The technique described in this protocol allows to screen for therapeutic molecules controlling T cell motility in tissues. The accessibility of the culture system permits to apply some pharmacological reagents and test their consequences on T cell migration. It is now accepted that in growing tumors, CD8 T cells exhibit a low migration and reduced contacts with tumor cells11. Importantly, the scarce presence of T cells in tumor cell regions is associated with a bad outcome and is considered to be one of the resistance mechanisms to T cell-based cancer immunotherapy. Multiple obstacles limiting T cells from migrating in tumors have been identified including a dense extracellular matrix. In this respect, the identification of molecules able to restore T cell motility and interaction with malignant cells represents an important step in cancer immunotherapy. In a previous study, we found that a partial degradation of collagen with collagenase, acutely applied to human lung slices, improves the contact of T cells with tumor cells.
Finally, the possibility to keep human tumor slices in culture for up to several days7,12,13 offers a number of promising applications in term of T cells and immunotherapy. However, the stability of the model system during long term culture is an important parameter that needs to be thoroughly assessed.
Obtaining fresh and healthy tissue is a critical factor to produce quality slices with good immunostaining of CD8 cells, tumor cells, and stromal elements. Keeping the tumor biopsy at 4 °C before processing is crucial.
The handling of slices with fine forceps represents another critical step within this protocol. This should be performed with great care as the agarose can be easily damaged. A cut in the agarose will compromise the seal made by the stainless steel washer resulting in a leakage of the antibody containing solutions.
The authors have nothing to disclose.
We wish to thank Pierre Bourdoncle and Thomas Guilbert of the Cochin Imaging Facility (Institut Cochin, Paris) for advice and assistance with microscopes. This work was supported in part by grants from the French Ligue Nationale contre le Cancer, by the CARPEM (Cancer Research for Personalized Medicine), by the Fondation de France and by the European Union's Horizon 2020 research and innovation programme under grant agreement No 667980 (CARAT).
Upright confocal microscope | Leica | The use of a spinning disk confocal is recommended | |
Imaris software | Bitplane | This sofware is used for visualization of images and tracking of T cell migration | |
Vibratome | Leica | VT1200S | |
Fine Forceps | World Precision Instruments | 14142 | |
30 mm Culture inserts | Millipore | PICM0RG50 | |
Roswell Park Memorial Institute (RPMI) medium, GlutaMAX Supplement | ThermoFisher | 61870010 | Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/streptomycin |
Phosphate-buffered saline (PBS) | ThermoFisher | 20012019 | |
Hanks’ Balanced Salt Solution (HBSS) | Invitrogen | 14170088 | |
Low gelling temperature Agarose, type VII-A | Sigma-Aldrich | A0701 | |
Non-toxic butyl Cyanoacrylate Glue, Vetbond | 3M | 1469 | |
Slice anchors, 19.7 diameter, 2 mm spacing threads | Warner Instruments | 64-1415 | |
Stainless steel washers, 5 mm of inner diameter | Amazon | B004K1FDGQ | |
BV605-conjugated anti-CD8 (clone SK1) | BD Biosciences | 565289 | |
FITC-conjugated anti-EpCAM (clone HEA-125) | Miltenyi Biotec | 130-098-113 | |
BV510-conjugated anti-CD90 (clone 5E10) | Biolegend | 328125 | |
4',6-diamidino-2-phenylindole (DAPI) | ThermoFisher | D1306 |