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In Vivo PET Tracking of 89Zr-Labeled Vγ9Vδ2 T Cells to Mouse Xenograft Breast Tumors Activated with Liposomal Alendronate

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.


INTRODUCTION
Adoptive transfer of therapeutic T cells is a growing field in immunooncology, with spectacular clinical results against melanoma and hematological cancers. [1][2][3] Gammadelta-T (gd-T) cell therapy is one type of T cell therapy being explored, with recent data showing intra-tumoral gd-T cells are the single most favorable prognostic immune cell infiltrate. 4 gd-T cells perform roles belonging to both adaptive and innate immunity, playing a significant role in anti-infectious and anti-tumor immune surveillance. 5 Activated gd-T cells are highly cytotoxic, enhance the function of other immune cells, and act as antigen-presenting cells. 6 In humans, the Vg9Vd2 subtype of gd-T cells represents 1%-5% of circulating CD3 + T cells. 6 Their potent cytotox-icity and high proliferative capacity have made them candidates of choice for cancer immunotherapy. 7 The unique activation of Vg9Vd2 cells by phosphoantigens such as isopentenyl pyrophosphate (IPP) 8 allows them to discriminate between normal and metabolically disordered cells based on IPP expression levels. 9 The activation and targeting of gd-T cells to tumor tissue could, therefore, be improved by selectively increasing the presentation of phosphoantigens in cancer cells, for example, by using liposome-or nanocarrier-based formulations of aminobisphosphonate drugs (NBPs). 10 NBPs (e.g., pamidronate, alendronate, and zoledronate), 11 which increase the expression of IPP in target cells by inhibiting farnesyl diphosphate synthase, are hydrophilic molecules that accumulate in bone, but not in other tissues, and are rapidly cleared from the circulation. Encapsulating alendronate in liposomes has been shown to increase the therapeutic efficacy of gd-T cells in preclinical models. 12,13 Clinical studies of gd-T cell immunotherapy have shown a good safety profile and efficacy comparable to second-line anticancer therapies, but they have also highlighted the need for improvements. 14,15 Unknown aspects of adoptive gd-T cell therapy include their in vivo distribution and kinetics of arrival at the tumor site. Whole-body imaging is highly useful in this context by enabling in vivo tracking of administered cells. Many techniques exist for non-invasive cell tracking; [16][17][18] however, only nuclear imaging, and particularly positron emission tomography (PET), provides sensitive and quantitative, whole-body information with adequate spatiotemporal resolution. Hence, methods to radiolabel and track therapeutic cells using positron-emitting radionuclides are likely to become important tools for cell immunotherapy. 19 PET tracking of T cells has been performed with radiolabeled antibodies, antibody fragments, or lipophilic small molecules 20,21 and by reporter-gene imaging. 22 When genetic engineering is not required, e.g., for gd-T cells, a clinically applicable alternative to reporter-gene imaging is direct cell labeling with PET radionuclides. Immune cells have long been imaged clinically by single-photon emission computed tomography (SPECT) in this manner, for example, using [ 111 In]In(oxinate) 3 and [ 99m Tc]Tc-exametazime. 19 In this regard, the clinically approved 8-hydroxyquinoline (oxine) has been recently shown to be an excellent ionophore for cell labeling with 89 Zr (t 1/2 = 78.4 hr, b + = 22.3%). [23][24][25] However, to the best of our knowledge, no study has evaluated its use for tracking gd-T cells.
Here we report the first use of [ 89 Zr]Zr(oxinate) 4 for in vitro radiolabeling and in vivo tracking of human gd-T cells, including the effects of radiolabeling on gd-T cell functionality, proliferation, and DNA integrity. We applied this strategy in a xenograft model of breast cancer with an engineered cancer cell line that allows multimodal imaging to track tumor cells. A liposomal aminobisphosphonate was administered to increase T cell trafficking to the tumor.  Figure 1A). The radiochemical yield was 77.6% ± 11.8%  89 Zr retention by gd-T cells over 7 days after labeling with [ 89 Zr]Zr(oxinate) 4 (average incorporated activity: 34.3 ± 6.0 mBq/cell). Mean ± SEM of triplicate measures for 3 cell batches.
In Vitro Assays of 89 Zr-Radiolabeled gd-T Cells The purity of in vitro-expanded gd-T cells plateaued 13À15 days post-isolation ( Figure S3), at which point they were radiolabeled. Cells labeled with 6À20 mBq/cell proliferated similarly to unlabeled cells (p R 0.05; Figure 2A), while cells labeled with more than 50 mBq/cell ceased to proliferate in vitro, indicating a dose-dependent effect of 89 Zr on gd-T cell proliferation. A similar dose dependency was observed on gd-T cell death ( Figure 2B) and DNA damage, evaluated by the formation of gH2AX foci 26 1 hr after labeling ( Figures  2C and 2D).
To evaluate the cytotoxic ability of radiolabeled gd-T cells, we quantified the survival of MDA-MB-231.hNIS-GFP cancer cell monolayers. gd-T cells labeled with up to 600 mBq/cell showed no significant difference in cancer cell killing compared to unlabeled gd-T cells ( Figure 2E). As a control, adding 89 Zr up to 3 Bq/cancer cell in the medium was not toxic to cancer cells in the absence of gd-T cells. Even in 30-fold excess, gd-T cells showed no toxicity toward cancer cells in the absence of aminobisphosphonate ( Figure S4).
In Vivo PET Tracking of 89 Zr-Radiolabeled gd-T Cells 89 Zr-radiolabeled gd-T cells were administered intravenously in a mouse xenograft model of breast cancer followed by PET imaging Molecular Therapy at 1 hr, 48 hr, and 7 days after injection. We imaged the hNIS-expressing cancer cells by SPECT using 99m TcO 4 À . 27 We also evaluated the effect of PLA on gd-T cell homing to tumor sites. The study schedule is provided in Figure 3A.
The PLA dosing schedule was established using 111 In-labeled PLA, showing significant PLA tumor accumulation within 24-72 hr of administration ( Figure 3B; Table S1). The experimental group (PLA treated) received radiolabeled gd-T cells + PLA (5 mg/kg alendronate). Control groups (non-PLA treated) received radiolabeled gd-T cells with placebo liposomes, non-liposomal alendronate, or saline. An additional control group received gd-T cells killed by freeze-thawing to compare bio-distributions of viable and non-viable cells.
SPECT showed uptake of 99m TcO 4 À in tumors and endogenous NISexpressing organs (thyroid, salivary, and lacrimal glands and stomach; Figure 3C). At 1 hr after intravenous administration of 89 Zr-radiolabeled gd-T cells, PET revealed high amounts of radioactivity in the lungs in all groups, with signal also observed in the liver and spleen ( Figures 3C and 3D). There was significantly higher uptake in the liver in the ALD group versus the PLA group. At tumor sites, the 89 Zr signal was close to background ( Figure S5). After 48 hr, 89 Zr activity increased in the liver, spleen, and bones in all groups and decreased in the lungs. Uptake of 89 Zr was observed at the tumor site only in the PLA group ( Figure S5), suggesting the presence of radiolabeled gd-T cells. Importantly, this was significantly higher in PLA-treated animals compared to control animals treated with non-liposomal alendronate ( Figure 3D). Enlarged tumor views showed heterogeneity in tumor tissue, with live tissue, expressing a functional hNIS protein 18,27 and represented by a donut of 99m Tc signal surrounding a core of non-viable tumor cells  ( Figure 3E). 89 Zr signal in tumors was heterogeneous, with some co-localizing with 99m Tc at the edges and foci of 89 Zr signal inside the tumor. After 7 days, 89 Zr activity remained high in the liver; increased in the spleen, bones, and kidneys; and was indistinguishable from background in tumors. Uptake values are provided in Table S2. Compared to other treatment groups, PET images of killed gd-T cells showed a higher accumulation in the liver immediately after injection and increased uptake of 89 Zr in the kidneys at later time points ( Figure S6).
Ex Vivo Bio-distribution of 89 Zr-Radiolabeled gd-T Cells Ex vivo g-counting 7 days post-administration of radiolabeled cells revealed a high concentration of 89 Zr in the spleen (153.5% ± 88.8% injected dose [ID]/g averaged across all groups, N = 24) and liver (58.1% ± 10.6% ID/g, N = 24) in all groups, followed by lung and bone tissue ( Figure 4A). Uptake of 89 Zr in tumors from PLA-treated groups (2.1% ± 0.8% ID/g) was significantly higher than in non-PLA groups (1.2% ± 0.3% ID/g; Figure 4B), suggesting higher gd-T cell numbers in PLA-treated tumors. Bone uptake of 89 Zr in PLA-treated groups (6.5% ± 0.8% ID/g, N = 9) was significantly lower than in other groups (10.0% ± 1.1% ID/g, N = 12; p = 0.0238). Uptake in kidneys was significantly higher with killed gd-T cells than in other treatment groups (Table S3). Uptake in other organs showed no major differences between treatment groups.
Tumor section autoradiographs showed a strong signal originating from hNIS-accumulated 99m TcO 4 À . Autoradiography was repeated after 4 days to allow for the decay of 99m Tc and the capture 89 Zr signal.

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Sections from PLA-treated animals showed increased 89 Zr signal compared to non-PLA-treated animals. The 89 Zr signal was higher in the tumor periphery, whereas the 99m Tc signal was uniformly distributed ( Figure 4C Figure S7). These cells were also visible in the spleen and liver after 7 days, but not in kidney sections ( Figure 5D) or in control tissues of mice not administered gd-T cells ( Figure S8).

DISCUSSION
[ 89 Zr]Zr(oxinate) 4 synthesis has been reported previously by our group 23 and others. 24,25 The temperature-independent labeling efficiency of gd-T cells with [ 89 Zr]Zr(oxinate) 4 suggests this is a passive process, in line with results from Sato et al. 24 Sufficient radiotracer retention within cells is important to ensure that the imaging signal reflects labeled cells rather than free radiotracer bio-distribution. We observed an efflux of approximately half of the incorporated 89 Zr over 1 week in vitro, which we believe does not interfere with in vivo imaging within this time frame. Uptake of 89 Zr in the bone can be used to estimate the amount of tracer that leaked from the cells. 25,28 Retention of [ 89 Zr]Zr(oxinate) 4 is dependent on cell type, and our results are comparable to those observed with dendritic, bone marrow, and chimeric antigen receptor (CAR)-T cells. [23][24][25] Comparable levels of tracer efflux have been observed from lymphocytes labeled with [ 111 In]In(oxinate) 3 , 29,30 the current gold standard for cell tracking by nuclear imaging.
A radiotracer for cell tracking must not significantly alter the phenotype, survival, proliferation capacity, and functionality of labeled cells. We demonstrated that the effects of [ 89 Zr]Zr(oxinate) 4 on gd-T cell survival, proliferation capacity, and DNA damage were kept minimal for doses up to 20 mBq/cell but were significant at doses R50 mBq/cell. The cytotoxicity of radiolabeled gd-T cells However, the therapeutic efficacy of gd-T cells presumably also relies on their in vivo proliferation ability; hence, we suggest that radiolabeling gd-T cells with [ 89 Zr]Zr(oxinate) 4 should ideally not exceed 20 mBq/cell. This could lead to sensitivity issues on conventional PET scanners. Indeed, our experiments show that ex vivo gammacounting tumors could reveal amounts of 89 Zr indistinguishable from background in our PET imaging system at day 7. Assuming that a human cell-tracking study would require 37 MBq 89 Zr 31 and 10 9 gd-T cells, 14 this would equate to an average of 37 mBq/cell, which we have shown not to be excessively damaging to gd-T cells. Upcoming developments in PET technology, such as total-body PET, 32 should reduce the required 89 Zr activity per cell (by a factor of 40) and overcome these sensitivity issues.
For in vivo studies, a xenograft model of human breast cancer in immunocompromised mice 12,33 was chosen, as mice do not possess a subset of T cells functionally equivalent to human Vg9Vd2 T cells. 34 We tracked gd-T cells radiolabeled with [ 89 Zr]Zr(oxinate) 4 (30À300 mBq/cell) by PET 1 hr, 48 hr, and 7 days after intravenous injection. We simultaneously used 99m TcO 4 À to visualize hNIS-expressing tumors by SPECT. The in vivo distribution of 89 Zr-labeled gd-T cells over time was similar to that observed in studies of adoptively transferred gd-T 35,36 and other T cells. 25,37,38 89 Zr uptake was significantly increased in PLA-treated tumors, suggesting that PLA increases homing of these cells to the tumor site. Accumulation of gd-T cells at the tumor site 48 hr after administration was also observed by others. 35 Uptake values for the spleen and tumor determined by image-based quantification are lower than those determined by ex vivo bio-distribution. This can be explained by the small size of this organ and significant partial volume effect (spleen) and the liquid or necrotic tumor core that leaked upon dissection.
For instrument sensitivity reasons, some imaging studies were performed with higher doses of 89 Zr than recommended above. However, the distinctly different distribution pattern observed with killed gd-T cells suggests that radiolabeling with up to 300 mBq/cell, which preserved cytotoxic functionality in vitro over 48 4 . Cell concentrations during labeling and in vitro assays were in the range of 1À5 Â 10 6 /mL. In comparison, using in vitro 89 Zr retention values, cell concentrations extrapolated from PET-computed tomography (CT) images in the organs showing the strongest 89 Zr signal (spleen, liver, and lungs) would be in the range of 0.5À5 Â 10 6 cells/mL. We therefore expect the DNA damage sus-tained by gd-T cells, due to both self-irradiation and crossfire, after in vivo administration to be comparable to that observed in vitro.
Considering the strong affinity of the 89 Zr 4+ ion for bone, 28 the relatively low bone accumulation of 89 Zr indicates limited efflux of weakly chelated 89 Zr, and it suggests that 89 Zr is mostly retained by gd-T cells after injection. The lower accumulation of 89 Zr in the bones of PLAtreated animals compared to other groups also suggests reduced efflux of 89 Zr from gd-T cells after PLA treatment.
Histology confirmed the presence of gd-T cells in the tumors, spleen, and liver, using the CD3 marker. 40 Immunohistochemistry and autoradiography suggest that gd-T cells accumulated mostly at the periphery of the tumor. The small number of cells observed by immunohistochemistry precludes statistical comparison. Furthermore, these techniques can only image the solid portion of the tumor. PET imaging not only allowed visualization of the whole, intact tumors but additionally revealed heterogeneous distributions of 89 Zr in tumors, which would be challenging to observe by histology. Combined with the non-invasive nature of PET imaging, this further highlights the value of using PET tracers such as [ 89 Zr]Zr(oxinate) 4 for cell tracking. The high uptake of 89 Zr in the liver and spleen was mirrored by the large numbers of human CD3 + cells observed in these tissues, consistent with the bio-distribution of radiolabeled gd-T cells. In contrast, the apparent absence of CD3 + cells in the kidneys, despite higher 89 Zr uptake than in the tumor, and the fact that the kidney uptake of 89 Zr was significantly higher in animals administered killed gd-T cells than in other groups both suggest that the radioactivity detected in the kidneys corresponds to 89 Zr progressively released from gd-T cells in other organs. A limitation of directly labeling cells is that the radionuclide can leak out over time and be taken up by adjacent tissue. Although immunohistochemistry demonstrates the presence of the administered gd-T cells in the tumors, this technique cannot determine whether the 89 Zr signal originates from the gd-T cells or from in situ-labeled bystander cells.
A critical aspect of this type of cellular immunotherapy is that the therapeutic cells must be activated at the target site and reach the tumor in sufficient numbers. gd-T cell toxicity toward cancer cells is greatly amplified by bisphosphonates, suggesting a role for gd-T cells in the anti-cancer properties of bisphosphonates. 41 Here we sought to increase phosphoantigen expression in tumors by administering PLA, which delivers alendronate to the tumors in an untargeted fashion by virtue of the enhanced permeability and retention (EPR) effect. 42 Liposomal alendronate proved safer than other bisphosphonates and effective in potentiating gd-T cell therapy. 12,43 We have previously shown that the tumor-to-background uptake ratio of PLA increases over time and is significant after 3 days. 44 Here we observed that PLA administered 4 days in advance significantly increased the amount of 89 Zr reaching the tumor within 48 hr of radiolabeled gd-T cell administration. Our results suggest that gd-T cells home to the tumor within 2 days and remain there for at least 5 days. This was not observed in any other treatment group, demonstrating the importance of encapsulating the aminobisphosphonate in a tumor-targeting vehicle.
Clinical imaging studies of therapeutic T cells with [ 111 In]In (oxinate) 3 have been performed by radiolabeling only a fraction of the total administered T cells, [45][46][47] although evidence exists that distributing the total activity over a larger number of cells better preserves their proliferative abilities. 48 Our results suggest that radiolabeling the entire batch of gd-T cells with [ 89 Zr]Zr(oxinate) 4 might be the preferable option to avoid imaging excessively damaged cells. In two notable studies, g-scintigraphy revealed T cell uptake in tumors using only 1À3 mBq 111 In per cell. 46 4 can now be applied to answer fundamental questions in the preclinical and clinical development of gd-T cell therapies, e.g., whether the accumulation of gd-T cells at the tumor site or their distribution within the tumor correlates with therapeutic efficacy. Due to numerous molecular and cellular differences, the distribution of human gd-T cells in an immunocompromised mouse model cannot fully predict their behavior in a human host. However, the results of this proof-of-principle study can be used to design a clinical trial that will answer the question of the distribution of gd-T cells in humans after adoptive transfer.
Our results have implications for clinical translation, and they suggest using liposomal aminobisphosphonates as adjuncts to gd-T cell therapy. In the context of clinical protocols involving repeated infusions of gd-T cells, 15

Experiment Approval
Animals experiments were approved by the UK Home Office under The Animals (Scientific Procedures) Act (1986), PPL reference 7008879 (Protocol 6), with local approval from King's College London Research Ethics Committee (KCL-REC). Experiments using human T cells received approval from KCL-REC (Study Reference HR-16/17-3746). All donors provided written, informed consent.

Reagents, Animals, and Cells
Unless otherwise indicated, reagents were purchased from Sigma-Aldrich and Merck. Female SCID/beige (CB17.Cg-Prkdc scid Lyst bg-J /Crl) and Nod scid gamma (NSG) (NOD.Cg-Prkdc scid Il2rg tm1WjI /SzJ) mice (18À25 g, 10À20 weeks old) were obtained from Charles River (UK). gd-T cells were obtained as described previously, 12 using zoledronate (Novartis) and interleukin-2 (IL-2) (Novartis). Full details are provided in the Supplemental Materials and Methods. Population purity was assessed by flow cytometry (BD FACSCalibur), using pan-gd TCR (IMMU510, Beckman Coulter B49175) and anti-CD3 (OKT3, BioLegend 317307) monoclonal antibodies. Data were analyzed using Flowing version (v.)2.5.1 (http://flowingsoftware.btk. fi). Only batches with R80% gd-positive CD3 + cells were used for further experiments (R95% for in vivo experiments). MDA-MB-231.hNIS-GFP cells 27 were grown in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and L-glutamine (2 mM), and they were tested for mycoplasma contamination (e-Myco PCR detection kit, Bulldog Bio).  4 with an equivalent amount of DMSO was used as a control. After 10À30 min of incubation, cells were pelleted and the supernatants kept aside. The cells were washed with PBS, centrifuged, and the washings combined with the previous supernatants. The cells were suspended in growth medium or PBS for further experiments. Viability was assessed using the trypan blue dye exclusion method. Radioactivity in re-suspended cells and combined supernatants was measured in a gamma-counter. Cell-labeling efficiency (LE[%]) was calculated as follows.

PET Tracer Synthesis
LEð%Þ= activity of cell fraction activity of cell fraction + activity of combined supernatants For radiotracer retention and cell proliferation studies, radiolabeled (or vehicle-treated) gd-T cells were cultured as described above, and they were analyzed at various time points for viability (using trypan blue), determination of cell-associated radioactivity (by g-counting), and cell death (by flow cytometry using propidium iodide [PI]; Thermo Scientific). Further details are provided in the Supplemental Materials and Methods.

Cancer Cell-Killing Assay
MDA-MB-231.hNIS-GFP cells seeded in a 96-well plate at 10 4 cells/ well and incubated overnight were treated with 3 mM zoledronate or vehicle for 24 hr. The cells were washed and the medium was replaced with gd-T cells in growth medium. As a control for radiolabeled gd-T cells, an equal amount of 89 Zr in medium was added to some wells. After 48 hr, gd-T cells were removed by washing with PBS, and cancer cell viability was evaluated using the alamarBlue assay (Thermo Scientific), reading plates in a GloMax (Promega) reader (530 nm excitation and 590 nm emission filters).

Animals, Tumor Model, and Tumor Sensitization with Liposomal Alendronate
Approximately 1.5 Â 10 6 MDA-MB-231.hNIS-GFP cells were injected subcutaneously in the mammary fat pad between the fourth and fifth nipples in the left flank; tumors were grown over 3 weeks. Animals were randomly assigned to experimental groups, and investigators were not blinded to cohort allocation when assessing outcomes. Cohort sizes were chosen based on prior experience, 44,50 in compliance with local regulations concerning animal experiments. Liposomal formulations were prepared at Shaare Zedek MC as previously described. 13 Alendronate-loaded liposomes (PLA) contained 1.5À5.4 mg/mL alendronate and 36À40 mmol/mL phospholipids. Placebo liposomes contained 20À50 mmol/mL phospholipids. PLA was co-injected with placebo liposomes for a total dose of 5 mg/kg alendronate and 4 mmol phospholipids per mouse in PLA-treated animals. Placebo-treated animals received empty liposomes corresponding to 4 mmol phospholipids per mouse. Another control group received 5 mg/kg alendronate (ALD). Formulations were injected intravenously (i.v.) 4 days before the administration of radiolabeled gd-T cells.
In Vivo PET and SPECT Imaging of gd-T Cells, Tumors, and PLA 89 Zr-radiolabeled gd-T cells (10 7 cells/animal in 100 mL, 0.3À3 MBq 89 Zr, single gd-T donor per experiment) were injected i.v. at t = 0 hr and imaged by PET/CT within 30 min. PET/CT imaging was performed for 30À240 min (as indicated) on a nanoScan PET-CT scanner (Mediso  3 and administered i.v. (7 MBq 111 In/mouse) to NSG mice. PET-and SPECT-CT datasets were reconstructed using a Monte Carlo-based full-3D iterative algorithm (Tera-Tomo, Mediso). Images were co-registered and analyzed using VivoQuant v.2.50 (Invicro). Regions of interest (ROIs) were delineated for PET activity quantification in specific organs. Uptake in each ROI was expressed as a percentage of injected dose per volume (% ID/mL).

Ex Vivo Bio-distribution Studies
Mice from imaging studies were used for bio-distribution studies on day 2 or 7. After culling, organs were dissected, weighed, and g-counted together with standards prepared from a sample of injected material. The percentage of injected dose per gram (% ID/g) of tissue was calculated. Organs were cryopreserved in optimal cutting temperature (OCT) compound (VWR) for autoradiography and/or formalin fixed and paraffin embedded (FFPE) for histologic analysis.

Autoradiography
Cryopreserved tissues were cut (50 mm), mounted on poly-L-lysinecoated slides (VWR), fixed in 4% paraformaldehyde (PFA), mounted in Mowiol, and exposed to a storage phosphor screen for 20 min at 3 hr post-dissection to obtain the 99m Tc signal, then for 48 hr at 4 days post-dissection to obtain the 89 Zr signal. The storage phosphor screen was read using a Cyclone Plus imager (PerkinElmer), and images were processed with ImageJ.

Immunohistochemistry
Briefly, FFPE organ blocks were sliced and stained using a Discovery