1. Mouse model
Two animal models were used to obtain the ideal strain for this work. Mice with transgenic bacterial artificial chromosome FoxP3.LuciDTR were mated with the inducible animal model of intestinal cancer Lrig1CreERT2/+; Apcflox/+ animals and thus generate animals with the phenotyping of interest which is Lrig1CreERT2/+; Apcflox/+; FoxP3.LuciDTR.
We used both male and female mice and the inductions were performed between 6 to 8 weeks of age. All mice were housed in a pathogen-free facility with a rigorously regulated light cycle (12 hours of light/dark) and were fed standard rodent chow and given water ad libitum.
a. Lrig1CreERT2/+; Apcflox/+
The heterozygous Lrig1CreERT2/+; Apcflox/+ mouse is an inducible model of intestinal cancer. Tamoxifen-inducible Cre (CreERT2) is targeted to the translation site of the endogenous leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1) - positive (Lrig1+) locus. These animals undergo Cre-Lox recombination in exon 14 of the Apc (Adenomatous Polyposis Coli) gene, leading to a frameshift mutation in codon 580, leading to an interruption of Apc function (Figure 5). These cells are considered quiescent intestinal stem cells that have in their crypts leucine-rich repeats and
Figure 5 - Scheme of deletion of the Apc floxed allele in Cre recombinase and in quiescent intestinal progenitor cells expressing Lrig1 occurs due to tamoxifen-induced Cre-mediated recombination.
Adapted from Powell et al., 2014.
31 immunoglobulin-like domains 1 (Lrig1) as a marker. Loss of the Apc gene in these cells triggers multiple intestinal lesions [68]. When the Cre-ERT2 fusion protein binds to the estrogen tamoxifen (4-hydroxy tamoxifen - OTH), it becomes active [69]. The heterozygosity present for CreERT2 and Apc flox ensures the expression of Lrig1 and that the hit on the second allele of the apc gene occurs later.
b. FoxP3.LuciDTR
FoxP3.LuciDTR mice were developed by combining cDNAs for enhanced green fluorescent protein (eGFP), CBGr99 luciferase and human diphtheria toxin receptor (DTR). The construct was placed at the start codon (ATG) in the third exon of the foxp3 gene in BAC using Escherichia coli and then inserted into the oocytes of fertilized C57BL/6 mice heterozygous for eGFP-DTR-Luciferase [70]. DTR is expressed with GFP-encoding sequences linked to the FoxP3 locus (Figure 6). The depletion mechanism works with the administration of diphtheria toxin (DT) that binds to its receptor. This animal model allows, in addition to the ablation of cells, the observation of the population through the bioluminescence emitted due to the presence of luciferase in the presence of its substrate, D-luciferin. The eGFP excitation also allows an observation within the visible spectrum.
Figure 6 - Construct for generation of BAC transgenic Foxp3.eGFP- 2A-DTR-2A-Luciferase (Foxp3.LuciDTR) mice. Adapted from Suffner et al., 2010.
32 c. Lrig1CreERT2/+; Apcflox/+; FoxP3.LuciDTR
This animal model was generated after crossing the two animals mentioned above in the institute’s animal house. Upon reaching maturity, this strain underwent tumor induction and regulatory T cell ablation procedures to respond to the purpose of this study. To work with the animals, it was necessary to take an animal experimentation course to comply with the experimental protocol approved by the National Authority for Animal Health (DGAV). All animal studies were conducted with the authorization of the local animal ethics committee and in compliance with European Union directives and Portuguese legislation.
2. Tumor induction
Tumor induction in Lrig1CreERT2/+; Apcflox/+; FoxP3.LuciDTR animals occurred with 100µL of tamoxifen injection and was performed for three consecutive days through intraperitoneal administration. We added 100mg of tamoxifen (Sigma Aldrich: T-5648) into pre-heated 5ml of corn oil (Sigma Aldrich C-8267), obtaining 20mg/ml final concentration of tamoxifen solution and kept for 24 hours in a thermos agitator at 37°C.
Storage was in a refrigerator at 4°C. To confirm whether the animals’ developed tumors, we performed a colonoscopy on day 50 after induction using The Mainz Coloview mini- endoscopic system (KARK STORZ, Tuttlingen, Germany). On days 70 and 90 after induction, some animals received diphtheria toxin (DT, Unnicked, Corynebacterium
Figure 7 - Scheme of tumor induction procedure by tamoxifen for 3 consecutive days, Tregs depletion by DT on days 70 and 90 after inductions. Tumor proofing and depletion assays were done once to confirm the success of the assays. Colonoscopy on day 50 and optical analysis on days 90 to 104. Part of the animals were euthanized at 120 days post induction, another part remained for survival, being euthanized when moribund. After euthanasia, tissues were harvested for analysis. Scheme made in Biorender.
33 diphtheriae – Calbiochem (322326-1MG), while others received saline solution as our control. We diluted the 10 µl DT solution (1mg/ml) by adding 990 µl of sterile PBS to have a final concentration of 0.01 µg/µl. following the protocol, when performing 1 injection use 1 µg per dose, so we injected 100ul of DT per animal. Blood samples were collected 24 hours after second DT injection for analysis by flow cytometry and observation of Treg cell depletion. The collected blood was inserted into a K3 EDTA tube (AQUISEL®, Barcelona, Spain), which contains an anticoagulant substance.
Bioluminescence visualization through luciferase and eGFP using in vivo imaging system (IVIS®) was performed on days 90, 91, 97 and 104 (2 weeks) to observe the cell population of Tregs full body cells. hair shaving was waived for analysis by IVIS. All animals were euthanized at 120 post-induction or as soon as they presented symptoms of anemia, weight loss, rectal prolapse by asphyxiation in CO2 chamber and cervical dislocation. We then collected tumors, lymph nodes (mesenteric and inguinal) and spleen for histology and flow cytometry (Figure 7).
3. Collection of cells from tumor, LNs and Spleen
Tumors collected from both mouse groups were cut into small pieces and then digested with collagenase IV solution (90% RPMI 1640 with L-Glutamine medium, 10%
FBS, 1mM CaCl2, 1mm MgCl2, and 100U/ml collagenase IV) for 30 minutes and 37°C.
The samples containing digested tissues are then passed through a cell strainer and washed with NaCl to extract a cell-rich suspension. In addition to the tumor harvest, the spleen and lymph nodes were also collected, which were also digested by passing through a cell strainer and resuspended in Red Blood Cell Lysis (90% NH4Cl and 10%
Tris-HCl). After obtaining the cell suspension, the samples were centrifuged for 5 minutes at a speed of 1700 rpm (or 611 g-force in Eppendorf® Centrifuge 5810R) and 4°C and then resuspended in FACS buffer (PBS and 10% FBS) or freezing medium (FBS with 10% DMSO) when frozen at -80°C. The area of the tumors was obtained through the measurement performed using a digital caliper. Tumors were measured in two dimensions, transverse (width) and longitudinal (length) diameter. For elliptical tumors the formula is 𝐴𝑒= (𝑊
2 ×𝐿
2× 𝜋), for circular tumors the length and width are equal, so 𝐴𝑐 = 𝐿2× 𝜋.
34 4. Immunohistochemistry assay
The tumors collected from the animals were also destined for immunohistochemistry and hematoxylin and eosin staining (H&E). The samples were immersed in 10% formalin for 24 hours. Tissue processing was carried out after 24 hours, with cassettes and tissues immersed in a series of solutions: 70% ethanol, 80% ethanol, 90% ethanol, 2 consecutive rounds 100% ethanol, 3 times in Clear Rite for 1 hour each, and 2 rounds in paraffin for 1 hour each. Then, the samples were immersed in paraffin wax to be sectioned using a microtome to be ready for the immunohistochemistry assay. In the middle of the assay, the primary antibodies used for the incubation were anti-FoxP3 (ab215206, Abcam), anti-CD3 (ab16669, Abcam), and anti-CD4 (ab183685, Abcam). To analyze tissue histology, H&E staining was performed. After completion of this assay, the slides are ready to be analyzed by optical microscopy.
5. Immunophenotyping of T cells
The immunophenotyping assay was performed for cells collected from tumors, spleen, and lymph nodes of Lrig1CreERT2/+; Apcflox/+; FoxP3.LuciDTR mice. Cells were stained with a mix of anti-CD3 (APC - 2297341- Invitrogen), anti-CD4 (PerCP- 2246965 - Invitrogen), anti-CD25 (PE-Cy7 - 2386301 - Invitrogen) monoclonal antibodies plus a fixable live/dead dye Zombie (APC-Cy7 – 423105/423106 - BioLegend). For analysis, the FACS CANTOTM II cytometer was used. To deepen the FACS results, the FlowJo software was used.
6. Statistical Analysis
Statistical significance of tumor size was analyzed using Kruskal-Wallis and Dunn's test and unpaired T test. The number of lesions was analyzed using Mann-Whitney test.
Due to the small number of events from each timepoint some statistical analysis has low reliability. The statistical analysis was performed using GraphPad Prism® 8 software (GraphPad Inc.) *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
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36
4. Results
1. Impact of tumor-infiltrating Tregs in an animal model of intestinal carcinogenesis
The Lrig1CreERT2/+;Apcflox/+mouse model was chosen as an intestinal neoplasia model, where the loss of one copy of the Apc allele is mediated by Cre recombinase activity under the control of leucine-rich repeats and immunoglobulin protein 1 (Lrig1) gene promoter, and upon treatment with of tamoxifen by intraperitoneal injection. As a consequence of Apc loss, these mice develop multiple tumors throughout the intestinal mucosa. Some of the benefits of using this animal model is the fact that it is already well characterized, and that the timing of cell transformation and tumor development is induced by tamoxifen treatment. Finally, the ease of intestine isolation and lesion individualization and collection allows practical and accessible work methods. The other animal model used by us was Foxp3.LuciDTR, which express an enhanced GFP, luciferase and the diphtheria toxin (DT) receptor in the Foxp3 gene. This model allows the depletion of Treg cells in a practical way with DT injection and the visualization of Treg cells through bioluminescence via luciferase detection assays. We pooled and mated the Lrig1CreERT2/+;Apcflox/+ with Foxp3.LuciDTR mice in order to generate our strain of interest: Lrig1CreERT2/+;Apcflox/+;Foxp3.LuciDTR. Thus, in this new mouse model we were able to induce intestinal tumors with or without the ablation of Treg cells to address the impact of Tregs on intestinal tumor formation and development. Intestinal lesions found along the intestinal mucosa of these animals were collected to study the behavior of regulatory T cells (Tregs) in a tumorigenic niche, how they may influence tumor growth by regulating the immune response, and if the ablation of their functions can play an immunotherapeutic role.
First, we proceeded to the characterization of the animal model Lrig1CreERT2/+; Apcflox/+, which consisted in the quantification of the tumor number and area, developed along the intestinal tract. Afterwards animals were injected with tamoxifen for three consecutive days between 6 to 8 weeks of age and euthanized 120 days later. The control group established by us are non-treated animals, which received injection of saline solution instead of tamoxifen. Colonoscopy was performed on day 50 after tamoxifen injection, which already revealed the presence of small lesions in the colon of some tamoxifen-treated mice (Figure 8), while all tamoxifen non-treated animals were devoid of tumors. After euthanasia, we counted the number of lesions present along the small intestine and colon and measured their sizes to obtain the area of each lesion. For
37 each animal, intestinal lesions, spleen, and lymph nodes were harvested and either processed as formalin-fixed paraffin-embedded (FFPE) samples for later use in histology and immunohistochemistry. Also, part of tumors, spleen and lymph nodes tissue were harvested in PBS and then digested and frozen in FBS (10% DMSO) for further flow cytometry assays.
For lesions quantification purposes, we divided and cut the small intestine in three equal-sized regions: proximal, intermediate, and distal (Figure 9).We observed that the mean number of tumors in the proximal region of the small intestine is higher in relation to the other regions, including the cecum and colon (p<0.0001) (Figure 10).
A B
*
* *
Figure 8 - Colonoscopy images of a Lrig1Cre/;Apcfl/+ mouse on day 50 after tamoxifen induction. (A) Macroscopic lesions are indicated by asterisks; (B) healthy intestinal tract, without visible lesions.
Figure 9 - Representative scheme of divisions of intestinal regions after biopsy. (A) Proximal region of the small intestine; (B) Intermediate region; (C) distal region; (D) Cecum and part of the colon.
38 From a total of 31 mice, 26 presented macroscopic lesions in the proximal region. 5 animals presented lesions in the intermediate zone, and 6 animals in the distal region.
For colon and cecum, which we pooled together because of the low number of lesions in each one of these regions, 12 animals showed the presence of neoplasia’s.
We also observed that tumors from the proximal region of the small intestine were the largest, compared to the intermediate and distal part of the small intestine (p<0.05), although a tendency for large tumors was also apparent for the colon and cecum counterpart.
The characterization of the animal model in study was followed by the identification of the infiltrating immune composition of tumor and normal intestinal tissue. Using flow cytometry analysis, we quantified the numbers and percentages of CD4+, CD8+ and Tregs (FoxP3+CD25+) T cell populations (Figure 11). Tumor tissues showed significantly higher percentages of CD4+ T cells than normal intestinal tissue (p<0.0001). The infiltration of Tregs was superior in tumor compared to normal tissue (p<0.001). As for CD8+ T cells, there seems to exist a tendency for a predominance of such cells in tumor samples compared to normal tissue, but with no statistical significance. In summary, in our mouse model we observed increased infiltration of immune cells are in intestinal tumors, when compared with normal intestinal tissue.
A B
Figure 10 - Tumor distribution in the small intestine and cecum and colon in Lrig1Cre/+;Apcfl/+animals. (A) Number of lesions found in each segment of the intestinal tract.
Each point represents a different animal (n=31); (B) Size of the lesions in mm2 in each region of the intestine. Each dot represents one lesion in a total of 10 animals. For statistical comparisons between groups, the Kruskal-Wallis test with Dunn's multiple comparison was used. * p<0.05; **** p<0.0001.
39
2. Impact of Treg depletion
Following the characterization and better understanding of the animal model, we proceeded with the ablation of Tregs by diphtheria toxin injection on days 70 and 90 after tamoxifen-driven tumor induction. To confirms depletion of Tregs, we collected a blood sample from an animal 24 hours after the second injection and compared with a blood sample from an untreated mouse. Flow cytometry revealed fewer Treg cells in the treated animal compared to the untreated animal (Figure 12).
FoxP3 FoxP3
FSC
A B
****
A B C
***
Figure 11 - Immune composition of tumor tissue samples (n=12) and normal tissue (n=10) from Lrig1Cre/;Apcfl/+ mice. Each point corresponds to pool analysis of tumors/normal tissue taken from one animal (A) Percentage of CD4+ T cells; (B) percentage of CD8+ T cells (C) percentage of CD25+FoxP3+ cells (Tregs). For statistical comparisons, the Mann Whitney test was used. ***p<0.001; **** p<0.0001.
Figure 12 - FACS analysis of FoxP3 cells from peripheral blood samples between non-treated and DT-treated animals 24h after the second injection with diphtheria toxin. (A) Gate for FoxP3 population in non-treated animals; (B) population gate of FoxP3 in DT-treated animals.
40 In parallel with flow cytometry, we used in vivo imaging system (IVIS) as a second method to confirm that Treg depletion was effective. This experiment was possible due to the presence of a bacterial artificial chromosomes (BAC) in our transgenic mouse model. Briefly, the BAC has a construct composed of cDNAs for eGFP, human DT receptor and luciferase, which was inserted into the third exon of the Foxp3 gene.
Therefore, to observe the Tregs in mice, we target Foxp3+ cells, as it is Tregs transcription factor and allows the use of luciferase to obtain bioluminescence. We injected the mice with the luciferase substrate, D-Luciferin, and after 10 minutes we observed the bioluminescence of Treg cells (Figure 13). This procedure was performed on day 0 (D0) when the treated animals received the 2nd dose of DT, which corresponds to day 90 after tumor induction. The detected bioluminescence was translated into Relative Lights Unit (RLU), and the higher the bioluminescence the higher the RLU values. Untreated animals also received D-luciferin and served as our control. The RLU obtained in D0 was approximately 60% in the whole body of the DT-treated animal.
A
B
Figure 13 - Visualization of Tregs depletion by Bioluminescence Imaging (BLI). (A) Full body BLI from Lrig1Cre/;Apcfl/+;FoxP3.LuciDTR mice non-treated and DT-treated. Images obtained after 2 doses of DT on day 0, day 1, day 7 and day 14. (B) Quantification of BLI signals from the DT-treated mouse shown in A.
41 When compared with the bioluminescence of the control animal; the emissions were much lower in DT-treated mouse. These results were expected, since the treated mouse had already received the second dose of DT, which reflects a reduction in bioluminescence to an RLU below 25% on day 1. We observed again on day 7 (D7) and on day 14 (D14) and the full body bioluminescence detected between these two days was approximately 50% RLU, being higher than on day 1 (D1).
After euthanasia, we assessed the bioluminescence of the dissected small intestine with eGFP excitation emission, which was excited at a wavelength of 488 nm and the detected signal was at 510 nm (Figure 14). The small intestine of mice treated with DT exhibited decreased levels of bioluminescence when compared to the small intestine of vehicle-treated mice, injected only with saline solution. These trials show that Treg depletion occurred partially with success after DT administration.
3. Ablation of Tregs results in decreased number of tumors in Lrig1CreERT2/+;Apcflox/+; Foxp3.LuciDTR mice.
In order to investigate the impact of Treg cell ablation in disease development and progression in our mouse model, we used two different endpoints, early and late (survival) endpoints. The early endpoint was set at 120 days (n=6) and the late endpoint, or survival (n=8), was set as a humane endpoint, determined as irreversible signs of
A B
Figure 14 - BLI of isolated intestines. (A) Small intestine of untreated animal with high BLI; (B) Small intestine of DT-treated animal with low signal emission.
42 disease manifestation, such as severe weight loss, presence of rectal prolapse, bloody stools and/or anemia. For both endpoints, we used untreated mice, which received only saline solution injection, and treated mice, in which DT was administered.
We observed that for the survival analysis, treated animals had a longer survival time than the untreated ones (Figure 15). We also observed that three animals in the DT- treated cohort had a survival time of approximately 350 days, similar to two untreated animals. Although these results were not statistically significant, we were able to notice a tendency for treated animals to survive longer than the untreated counterpart.
For both early and late endpoint groups, we counted, measured the size, and collected the tumors from the different regions of the small intestine and from colon and cecum. The average number of lesions in the intestinal tract was higher in the non- treated group with early endpoint animals (p<0.01). For statistical purposes, we pooled the total number of lesions along the different regions of the intestinal tract. (Figure 16A).
As for the late endpoint (survival), the average number of lesions appears to be similar for treated and untreated animals (Figure 16B).
Figure 15 - Survival graph of DT-treated (red) and untreated (blue) animals from the late endpoint group.