The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 µl blood sample is also described.
The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 µl whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.
Malignant mesothelioma (MM) is an aggressive malignancy which arises from transformed cells in the membrane (mesothelium) that lines the lung and abdominal cavities, heart and internal reproductive organs, and is the most common primary tumor of the lung cavity or pleura1,2. Exposure to asbestos fibres accounts for 80% of all MM, and while bans on asbestos use were introduced decades ago in most western countries, its widespread use in the community has left a deadly legacy. The World Health Organization has estimated that 107,000 people worldwide die each year from asbestos related diseases, with mortality rates continuing to increase. A new non-occupational incidence wave is also emerging and there is little understanding of when, and at what level this will peak3.
The majority of people with MM are diagnosed late when systemic chemotherapy represents one of the only viable options4. The most effective chemotherapy and current ‘standard of care’ (pemetrexed together with cisplatin5) was identified over 10 years ago. However failure of this treatment is inevitable and there are no proven second line options, leaving patients with a grim prognosis and median survival of only 12 months2. Therefore, there is an urgent unmet need for more effective treatments. Despite the examination of a number of novel therapies in clinical trials none has resulted in changes in practice. This is due in part to the low (5%) transference of pre-clinical results, generally performed in xenograft mouse models, to the clinical setting6-8. Such models do not faithfully recapitulate the complex aspects of the tumor microenvironment occurring in non-physiological locations, frequently in the absence of a functioning immune system9.
Syngeneic orthotopic models create a significantly more realistic tumor environment than the commonly used subcutaneous xenograft models as the tumors occur in the correct physiological location with an intact immune system10,11. The larger size of the rat enhances its use as a rodent disease model, especially in drug studies where serial blood draws are required to assess treatment response and toxicity12. Furthermore, in models in which monitoring disease progression is difficult due to the location of the tumors (such as in the pleural cavity), the ability to monitor disease progression using factors found in the circulation is extremely attractive. The generation of a syngeneic orthotopic model of pleural mesothelioma using immune-competent rats is described. In addition, an easy and relatively non-invasive method for monitoring pleural disease progression by measuring circulating immune cells is also described.
All procedures involving animals were carried out in accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The protocol for this study was approved by the Royal North Shore Hospital Animal Care and Ethics Committee. Female Fischer 344 rats (F344, 150-200 g) were maintained at the Kearns Facility, Kolling Institute under standard conditions (12 hr light/dark cycles and free access to food and water).
Note: A flow chart for all experimental procedures is presented in Figure 1.
1. Preparation of Cells for Implantation
2. In vivo Implantation of Cells
3. Tail Vein Blood Collection
4. Sample Preparation for Immune Cell Profiling Using the Bead-based Method
Note: This single platform method relies on using commercially available absolute counting tubes that have a known number of beads for each sample. These tubes contain lyophilized pellets that dissolve during sample preparation, releasing the beads. The beads are fluorescently labelled and by gating on the bead population, absolute counts can be calculated.
5. Flow Cytometric Processing of Samples
Note: Perform on a 4 color flow cytometer.
6. Immune Cell Analysis
Note: Gating strategies and Boolean algebra are used to define each cell population. Boolean algebra is a logic based analysis method that allows for multiple operations in a single definition. The analysis software of the flow cytometer (e.g., BD CELLQuest) allows for the use of Boolean algebra. The equations are used to actively account for the significant negative reactivity that assists in defining the cell to more specifically identify each cell population. ‘Regions’ are used to define a ‘gate’. Regions define a 2 dimensional space whereas gates can be composed of numerous regions connected by algebraic operators (+, *,-, defined in Table 2).
The method used in this paper for the generation of an orthotopic model of pleural mesothelioma using II-45 cells resulted in animals succumbing to mesothelioma in a reproducible and rapid timeframe, with no rats dying due to the implantation method. Titration of the number of cells implanted determined that 1x 103 cells was the minimum number required for a fully penetrant model (100% engraftment). The different number of cells implanted in the rats changed the time course of the disease without appearing to affect the severity of the disease. Animals that received 1 x 103, 1 x 104 and 5 x 105 cells reached ethically defined endpoints by day 40, 30 and day 20 respectively (Figure 6).
Blood was collected from rats approximately twice each week with no complications observed related to the collection. It is important to note that no more than 10% of the total blood volume (or approximately 2 ml) should be collected in a 2 week period. A flow cytometric assay which combined gating strategies and Boolean algebra was established to identify lymphocytes, monocytes and neutrophils and the lymphocyte subsets T, CD4 T, CD8 T, B and NK cells. The mean and range for each cell type was established using control rats (n=5) and was compared to the mean and range of II-45 implanted rats at endpoint (Table 3). Endpoint values were first compared to determine whether differences existed in immune cell counts between healthy and diseased animals. In comparison to healthy control rats, total lymphocytes, CD4 T cells, B lymphocytes and NK cells decreased in II-45 implanted rats at endpoint whereas monocytes, neutrophils and neutrophil to lymphocyte ratio (NLR) increased.
To determine whether these immune cell parameters also changed with disease progression and thus could be used as surrogate markers for monitoring disease, longitudinal samples were also compared (Figure 7). The most informative longitudinal parameter was NLR with increases detected up to 7 days before ethical endpoints (Figure 7A). For rats with high dose cells, high NLRs were detected between day 7 and day 12 post implantation, whereas for rats with low dose cells, the NLR began to increase between day 18 and 22. Total lymphocyte counts decreased with disease progression in rats with a longer disease time course model (low cell dose: 1 x104, Figure 7B). This decrease was not evident in rats with a rapid time course (high cell dose: 5 x105). Monocytes remained at normal levels until the terminal collection when grossly increased counts were often observed (Figure 7C). The other immune cell counts (neutrophils, NK, B cell and CD4 and CD8 T cells) were more variable and thus less informative.
Figure 1. Flow chart of experimental procedure. Please click here to view a larger version of this figure.
Figure 2. Location and site of implantation. The rat is first anesthetized, placed on its back and the right regio costalis (chest) area is shaved to remove fur. On the right side, the 2nd nipple from the top is identified and the injection site is located 0.5 cm proximal to this, in between the 3rd and 4th rib from the bottom of rib cage (A). A spacer is then placed over the prepared needle and syringe to prevent the needle penetrating too deeply into the pleural cavity and 100 µl of cells or SFM is injected (B). Please click here to view a larger version of this figure.
Figure 3. Sampling technique for collecting blood from tail. Position a 23G x 1¼ needle parallel to the lateral vein (A). Slide the needle into the vein at a shallow angle, approximately 10 mm deep, and hold there for a few seconds until blood is visible (B) then remove needle. Collect blood using a pipette (C). If necessary gently stroke tail vein to ensure blood continues to flow. Pipette blood into a 0.5 ml EDTA collection tube (D), mixing as you go to prevent clotting. Please click here to view a larger version of this figure.
Figure 4. Labelling of blood sample in an absolute counting tube for flow cytometric analysis. Blood (25 µL) and antibodies are added to an absolute counting tube (A). The tube is then vortexed and incubated for 15 min at room temperature. Before the addition of ammonium chloride buffer to lyse the red blood cells, the sample appears turbid or opaque (B). Once lysed, the sample appears translucent and is ready for flow cytometry analysis (C). Please click here to view a larger version of this figure.
Figure 5. Gating strategy for flow cytometry analysis. A 6-dimensional dataset can identify lymphocytes, monocytes and neutrophils and the lymphocyte subsets T, CD4 T, CD8 T, B and NK cells. The defined gating strategies and algorithms incorporate both the negative and positive reactivity that define each cell type enabling specific identification. The number of beads in each absolute counting tube (found on the pouch for each tube) is recorded for each sample and is required for calculating the number of cells per μl blood for each subset as shown. The complete gating algorithm for each cell subset is listed to the right of the analysis plots.(Ai) Beads (R1) are initially counted by FITC (FL-1) versus APC (FL-4). Acquisition is set to approximately 10,000 events (9960 for this example). (Aii) Debris (R2) is identified by FSC versus SSC and removed from all subsequent gating. (Bi) Following removal of debris (R2) FSC versus SSC differentiates white blood cell population into lymphocytes (R3), monocytes (R4) and neutrophils (R5). (Bii) The cell populations identified in (Bi) are confirmed using the common leucocyte antigen CD45 gating on PE/Cy7 (FL-3) versus SSC. (Biii) The lymphocyte population (R3) is further differentiated into T cells (R6) using CD3 gating on APC (FL-4) versus SSC. (Biv) CD8 T cells are lymphocytes (R3) differentiated into T cell (R6) that also express CD8a (R9) and are gated on PE (FL-2) versus SSC. (Ci) B cells and NK cells are defined as lymphocytes (R3) which are CD3 negative. B cells (R7) are differentiated from NK cells as they express CD45RA and are gated by APC (FL-4) versus FITC (FL-1). (Cii) CD4 T cells are defined as lymphocytes (R3) that co-express CD3 (T cells, R6) and CD4 (R8) but do not express any of the other markers used. They are differentiated using FITC (FL-1) versus PE (FL-2) gating. NK cells are defined as any remaining lymphocyte that has not already been defined that also expresses CD161a. Please click here to view a larger version of this figure.
Figure 6. Survival curve for rats implanted with different doses of II-45 mesothelioma cells. Groups of rats were pleurally implanted with 5 x 105 (n=6), 1 x 104 (n=2), 1 x 103 (n=2) or 1 x 102 (n=4) II-45 mesothelioma cells in 100 µL volume and then monitored until ethical endpoint when they were euthanized. Survival was graphed using the log-rank (Mantel-Cox) Gehan-Breslow-Wilcoxon method.
Figure 7. Longitudinal circulating immune cell counts from rats with mesothelioma relative to the mean and range for healthy control rats. The healthy control mean for each cell type is shown as a horizontal unbroken black line (value is shown on the right Y-axis of each graph) and the range is depicted by the grey shaded area. The Y-axis shows the counts per μl of blood for the stated cell type, or the value for NLR. The X-axis is the time course for disease progression, where day 0 is the day of implantation. Longitudinal results for 2 rats implanted with 1 x 104 cells (A, B) and 2 rats implanted with 5 x 105 cells (C, D) are shown. (A) NLR, (B) total lymphocyte counts, (C) monocyte counts. Please click here to view a larger version of this figure.
Detector | Fluorophore | Details | Voltage | Mode |
FSC | — | — | E00 | Linear |
SSC | — | — | 450 | Linear |
FL-1 | FITC | 533/30 nm | 670 | Log |
FL-2 | PE | 585/40 nm | 675 | Log |
FL-3 | PE/Cy7 | 670nm (long pass) | 600 | Log |
FL-4 | APC | 675/25 nm | 735 | Log |
Table 1: Voltage detector settings for flow cytometric data acquisition using a 4 color flow cytometer. Amplitude gain is set at 1 for all detectors. FSC: forward scatter, SSC: side scatter, FITC: Fluorescein isothyocyanante, PE: Phycoerythrin, APC: Allophycocyanine.
Cell subset | Cell Markers | Gating Algorithm |
Beads | R1 | |
Debris | R2 | |
Cells (pan-leukocytes) | CD45+ | -R2 |
Lymphocytes | CD45+ FSC versus SSC | R3*-R2 |
Monocytes | CD45+ FSC versus SSC | R4*-R2 |
Neutrophils | CD45+ FSC versus SSC | R5*-R2 |
T cells (total) | CD45+/CD3+ | R6*R3*-R2 |
CD8 positive T cells | CD45+/CD3+/CD8a+ | R9*R6*R3*-R2 |
B cells | CD45+/CD3-/CD45RA+ | R7*R3*-R2 |
CD4 positive T cells | CD45+/CD3+/CD4+ | R8*R6*R3*-(R7*R3*-R2) |
Natural Killer cells | CD45+/CD3-/CD161a+ | R3*-[(R2+R6)+(R7*R3*-R2)] |
Table 2: Cell markers and gating algorithms to characterize each immune cell subset. For the gating algorithm, the ‘+’ symbol means ‘or’ and allows two separate populations to be added together even if their reactions do not overlap. The ‘-’ symbol means ‘not’ and can subtract one population from within another defined population or the reverse of a previously described population. The ‘*’ symbol means ‘and’ and defines a space that is present only when two reactions overlap.
Immune cell subsets | Control rats (n=5) | II-45 implanted rats at endpoint (n=4) | ||
Mean | Range | Mean | Range | |
Total lymphocytes | 165.7 | 111.5-207.0 | 97.9 | 54.4-137.0 |
CD4 T lymphocytes | 72.9 | 45.4-111.4 | 38.4 | 25.6-50.0 |
CD8 T lymphocytes | 36.6 | 26.0-48.3 | 32.1 | 16.1-46 |
B lymphocytes | 56.6 | 36.0-83.9 | 24.7 | 11.2-36.0 |
NK cells | 9.3 | 3.1-15.7 | 4.0 | 1.34-7.7 |
Monocytes | 27.1 | 12.7-39.0 | 373.3 | 42.4-575.8 |
Neutrophils | 51.4 | 24.3-87.0 | 159.2 | 51.1-366.0 |
NLR | 0.3 | 0.18-0.42 | 1.8 | 0.94-2.66 |
Table 3: The mean and range of immune cells in healthy control rats (n=5) and II-45 implanted rats at endpoint (n=4). Results per µl of blood are shown.
This paper details a method for the generation of a rat syngeneic orthotopic model of pleural mesothelioma and a simple method for monitoring disease progression through longitudinal blood sampling.
The II-45 model was developed by exposing Fischer 344 rats to asbestos fibers13. Although this exposure represents the true dynamics of host-asbestos-immune system interactions for mesothelioma pathogenesis, it has a long lag time (taking years to generate) and can be dangerous for the researchers due to the potential exposure to asbestos fibers. Orthotopic implantation of II-45 cells into the pleural cavity of animals provides a safe and rapid model of cancer progression which mimics the human disease in an immune-competent host11. However the exact methods and principles to adhere to during these experimental procedures are not well defined. The procedure described results in a highly reproducible model that is relatively independent of the experience of the operator. The critical steps are 1, harvesting II-45 cells in the exponential growth phase and accurate counting of viable cells; 2, correct positioning of the implantation site and ensuring the needle does not penetrate too deeply and puncture the lungs or damage the heart, and 3, maintaining and monitoring the well-being of the animals.
The II-45 cell line is fast growing and requires passaging every 3 to 4 days in culture, therefore when preparing for implantation, cells should be sub-cultured and growth monitored daily. Cells should be harvested and counted when in the exponential growth phase, i.e., at approximately 70% confluence. Resuspension of cells in serum free media rather than PBS reduces the stress on the cells while counting and preparing for implantation. Accurate cell counts are crucial for reproducibility, especially when dosing with low cell numbers (e.g., 100 to 1,000). The suspension should be homogeneous and consist of single cells (no clumps). An implantation volume of 100 µl enables accurate dosing without impairing lung expansion through excessive fluid in the pleural cavity. The key steps at implantation are correct positioning and limiting the needle penetration to a depth of no more than 12 mm. Correct positioning of the needle at the injection site is extremely important to ensure that the cells are implanted into the pleural cavity and not into the peritoneal cavity. Incorrectly implanting cells too low can result in mesothelioma tumors growing through the diaphragm, into the liver and peritoneal cavity. Restricting the penetration depth of the needle is also extremely important to prevent mortality as a result of internal organ damage. This restriction can be achieved through the use of a spacer over a long needle (as shown) or the use of a shorter needle with a shaft length of 5 mm to 12 mm. In our hands, no adverse events have occurred during cell implantation. It is also essential to replace the needle after every implantation. Failure to do so can result in tumors growing out of the chest cavity along the injection line. Such growth outside of the pleural cavity is due to residual cells on the needle shaft and may result in extended survival.
In our hands this model has a 100% tumor engraftment rate when administering ≥ 1 x 103 cells. Disease progression in the II-45 mesothelioma model is quite rapid, being less than 40 days when 1 x 104 cells are implanted and less than 20 days for a dose of 5 x 105 cells. This may limit the usefulness of this model for pre-clinical testing of some therapies. The II-45 cells, while not commercially available can be sourced with permission from the originator13. However, the same implantation techniques and principles may also be applied to other rat mesothelioma cell lines available from cell line repositories when matched with the relevant syngeneic rat strains. The optimal cell dose may vary with cell line and would need to be determined for each line.
Monitoring disease progression and the well-being of the animals is difficult as the tumors are internal and cannot be monitored by size. Therefore we sought to develop a method of monitoring the animals that could predict deterioration of their condition such as an increase in NLR or other change in immune cell markers before physical symptoms became visible. The blood screen developed uses only 25 µl of peripheral blood so that regular sampling can be performed with minimal distress to the animal. The sampling technique described requires practice to gain the necessary expertise. However, once mastered it is fast and minimally invasive.
A critical step in the blood analysis is to prevent clotting of the blood sample by rapid transfer to an EDTA tube. Clotted samples should be discarded as the counts will be inaccurate. Another critical step is the red cell lysis prior to cell analysis. If the red cells are not fully lysed, the increased background will falsely elevate the counts. While the initial setup of dot plots and gating is time consuming, once the settings and template are saved, the assay requires little adjustment. The gating strategy employed uses a multi-parameter Boolean algebraic approach to deconvolute the data. By using known combinations of exclusive and shared antigens the algorithms for each line of the gate strategy define the discrete cell type in this six-dimensional data matrix14. This coupled with established accuracy of the ratiometric bead-based technology15-17 has the advantage of enabling the enumeration of any defined cell type as both a proportion of cells and an absolute count per µl.
It is becoming increasingly recognized that the immune system plays an important role in the pathogenesis of cancer18. Both the failure of the immune system to recognize and destroy malignant cells and the suppression of immune cells by tumors can lead to uncontrolled tumor growth. Thus, the inclusion of an immune-competent tumor microenvironment is a critical consideration when modelling cancer and is a major advantage of syngeneic cancer models. These immune-competent models create a realistic and clinically relevant environment for tumor growth and immune interaction, which is deficient in xenogeneic models.
The immune parameters identified in this study that accompanied disease progression, i.e., lymphocyte counts, monocyte counts and the NLR, have also been shown to correlate with poor prognosis in human mesothelioma19-21 providing evidence for the relevance of the model and the blood test. In the models presented, the clinical utility of monitoring the immune parameters increased with the longer time course (lower dose) and may be further improved by more frequent sampling at later stages of the disease. While this paper focuses on a model of mesothelioma, the ability to monitor tumor progression through peripheral blood sampling can be utilized for all syngeneic cancer models. Further, the assay can also be used to assess the immunological response to different therapies. Previously we have used this assay to monitor the response to an anti-cancer vaccine therapy in a syngeneic glioma model (9L)22. In that study, CD4 and CD8 T cells were the most informative markers. We have also used this assay and the II-45 mesothelioma model to identify differences in the immune response to tumors resistant to different chemotherapeutics11. In that study, all parameters except CD8 T cells showed significant differences. The longitudinal monitoring during disease progression described here highlights the potential of different immune cell parameters to correlate with cancer progression and the well-being of animals when undertaking syngeneic orthotopic modelling of cancer in rats.
The authors have nothing to disclose.
The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.
EDTA Collection tube (0.5ml) | Greiner Bio One GmbH | 450480 | |
Rat T/B/NK Cell cocktail | BD Pharmingen | 558509 | anti-Rat CD3 APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78) |
anti-RAT CD8a PE | Biolegend | 200608 | (IgG1vClone G28) |
anti-Rat CD4 FITC (Domain 1) | Biolegend | 203406 | (IgG1 Clone OX-38) |
anti-Rat CD45 PE/Cy7 | Biolegend | 202214 | (IgG1 Clone OX-1) |
TruCount™ Tubes | Becton Dickinson | 340334 | Box of 50 absolute counting tubes |
RPMI 1640 media | Life Technologies | 11875-119 | |
foetal bovine serum (FBS) | Scientifix | FBS500-S (lot# 010101-1) | |
trypsin-EDTA | Life Technologies | 15400-054 | |
PBS tablets | Medicago AB | 09-9400-100 | |
23Gx1¼ Needle | Becton Dickinson | 302008 | |
1mL Syringe | Becton Dickinson | 302 100 | |
Fischer 344 Rat | Animal Resources Centre, Perth Australia | F344 | |
I.S.O (Isoflurane USP) | Veterinary Companys Australia (VCA) | B7058 | |
II-45 Rat Mesothelioma line | Zurich University | Note: The cell line was given as a gift and is not commercially available at the ATCC | |
FACSCalibur™ 4 colour | Becton Dickinson | 342975 | |
TRIS-HCL | SIGMA | T3253 | |
Ammonium Chloride | SIGMA | 9718 | |
Anaesthetic Machine (The stinger) | Advanced Anaesthesia specialists | #00449 |