In-Vivo Detection and Tracking of T Cells in Various Organs in a Melanoma Tumor Model by 19F-Fluorine MRS/MRI

Background 19F-MRI and 19F-MRS can identify specific cell types after in-vitro or in-vivo 19F-labeling. Knowledge on the potential to track in-vitro 19F-labeled immune cells in tumor models by 19F-MRI/MRS is scarce. Aim To study 19F-based MR techniques for in-vivo tracking of adoptively transferred immune cells after in-vitro 19F-labeling, i.e. to detect and monitor their migration non-invasively in melanoma-bearing mice. Methods Splenocytes (SP) were labeled in-vitro with a perfluorocarbon (PFC) and IV-injected into non-tumor bearing mice. In-vitro PFC-labeled ovalbumin (OVA)-specific T cells from the T cell receptor-transgenic line OT-1, activated with anti-CD3 and anti-CD28 antibodies (Tact) or OVA-peptide pulsed antigen presenting cells (TOVA-act), were injected into B16 OVA melanoma-bearing mice. The distribution of the 19F-labelled donor cells was determined in-vivo by 19F-MRI/MRS. In-vivo 19F-MRI/MRS results were confirmed by ex-vivo 19F-NMR and flow cytometry. Results SP, Tact, and TOVA-act were successfully PFC-labeled in-vitro yielding 3x1011-1.4x1012 19F-atoms/cell in the 3 groups. Adoptively transferred 19F-labeled SP, TOVA-act, and Tact were detected by coil-localized 19F-MRS in the chest, abdomen, and left flank in most animals (corresponding to lungs, livers, and spleens, respectively, with highest signal-to-noise for SP vs TOVA-act and Tact, p<0.009 for both). SP and Tact were successfully imaged by 19F-MRI (n = 3; liver). These in-vivo data were confirmed by ex-vivo high-resolution 19F-NMR-spectroscopy. By flow cytometric analysis, however, TOVA-act tended to be more abundant versus SP and Tact (liver: p = 0.1313; lungs: p = 0.1073; spleen: p = 0.109). Unlike 19F-MRI/MRS, flow cytometry also identified transferred immune cells (SP, Tact, and TOVA-act) in the tumors. Conclusion SP, Tact, and TOVA-act were successfully PFC-labeled in-vitro and detected in-vivo by non-invasive 19F-MRS/MRI in liver, lung, and spleen. The portion of 19F-labeled T cells in the adoptively transferred cell populations was insufficient for 19F-MRS/MRI detection in the tumor. While OVA-peptide-activated T cells (TOVA-act) showed highest infiltration into all organs, SP were detected more reliably by 19F-MRS/MRI, most likely explained by cell division of TOVA-act after injection, which dilutes the 19F content in the T cell-infiltrated organs. Non-dividing 19F-labeled cell species appear most promising to be tracked by 19F-MRS/MRI.


Introduction
Cell tracking by magnetic resonance imaging (MRI) is an emerging method to visualize and monitor labeled cells after transplantation non-invasively and without the use of ionizing radiation. Recently, 19 F-fluorine-MRI has been used to detect and track well-defined cell populations [1][2][3][4][5][6][7]. Because of the effective absence of 19 F background signal in the body, any 19 F signal detected after injection of a 19 F compound is unequivocally produced by this injected compound. As the MR signal is directly proportional to the amount of 19 F nuclei present in the tissue, it can be related to a reference of known 19 F concentration, rendering this technique quantitative [3,4]. Moreover, these compounds are not limited by signal decay over time and therefore the time window for their detection can last several days. Finally, the 19 F signal can be merged with conventional 1 H-MRI images to identify its exact anatomic location and to add information on structure, function, and tissue characteristics. Direct IV injection of emulsions containing 19 F-based perfluorocarbons (PFC) has been performed in different rodent models for angiography [8] and to detect non-invasively inflammation in myocardial infarction [5,9], cerebral ischemia [5], myocarditis [6], pneumonia [10], atherosclerosis [11], arthritis [12] and tumors infiltrated by macrophages [13]. Distinctively, defined cell populations such as dendritic cells [1], T cells [3,4,14,15], or mesenchymal stem cells [16] were tracked non-invasively in rodents by 19 F-MRI or 19 F-MR spectroscopy ( 19 F-MRS) after their in-vitro 19 F-labeling. Recently, clinical 19 F-MRI cell detection using labeling by PFC has also been described in patients with colorectal adenocarcinoma in order to detect autologous immunotherapeutic dendritic cells [7]. This technique could therefore be applied to detect tumor cells as well as to monitor adopted cell transfer cancer therapies.
In recent years adoptive cell transfer therapies using ex-vivo activated T cells have undergone intensive testing [17,18], and various types of T cells have been used for adoptive immunotherapy. It is essential to know whether the administered T cells reach their target and this is currently assessed by biopsies, which are invasive and not practical for all patients [18]. Also, with a biopsy-based approach the total amount of T cells in a tumor, their distribution, and the kinetics of cell fluxes are difficult to assess. Non-invasive visualization of the trafficking of administered T cells could potentially allow one to predict responsiveness to these therapies. Therefore, a reliable non-invasive imaging method to monitor anti-tumor cell traffic is highly desirable. Moreover, as T cells with specific anti-tumor properties can migrate to and infiltrate tumor tissue by recognizing tumor antigens [19], they could, in principle, be used as a probe to detect tumor cells at metastatic sites when labeled with PFCs.
In the present study the migratory behavior of 3 different cell populations was tracked by means of non-invasive 19 F-MRS and 19 F-MRI and compared with invasive flow cytometry analyses and high-resolution in-vitro 19 F-NMR. Initially, splenocytes (SP) were labeled in-vitro by a PFC to test the feasibility of non-invasive in-vivo tracking by 19 F-MRS and 19 F-MRI in control mice. SP represents a heterogeneous cell population comprising not only T cells (both CD8 + and CD4 + , naïve, effector, memory and regulatory cells), but also B cells and antigen presenting cells (including dendritic cells, monocytes, macrophages and myeloid cells). The activated T cell populations, T OVA-act and T act , whereas, are mostly CD8 + and these cytotoxic lymphocytes express one unique T cell receptor (TCR) called OT-1. To distinguish how the T cells were activated and expanded in-vitro, we named "T act " the T cells that were stimulated with anti-CD3 and anti-CD28 antibodies, and "T OVA-act " the cells derived from single-cell suspensions of dissociated spleens stimulated with the specific OVA 257-264 peptide. The OVA 257-264 antigen was used as a tumor-specific antigen in the current study, and T OVA-act and T act were produced from OT-1 mice expressing only the TCR OT-1 specific for K b -OVA 257-264 which is expressed at the surface of B16-OVA tumor grafted on recipient mice.
Splenic-derived OT-1 CD8 + T cells, stimulated either by OVA-peptide (= T OVA-act ) or by anti-CD3 and anti-CD28 antibodies (= T act ) will expand and differentiate into various states including central memory (T CM ), effector memory (T EM ) and terminally differentiated, shortlived effector T cells (T E ). Importantly, the newly activated T cells will also maintain a high state of proliferation for several days. While T E cells are typically found in peripheral tissue and provide a critical first line of defense to foreign antigen, T CM cells migrate to areas of secondary lymphoid organs, and compared to naive T N cells have a higher sensitivity to antigen stimulation. T EM tend to home to inflamed tissues, and have a more rapid effector function as compared to T CM [20]. Activated tumor-antigen specific T OVA-act and T act were labeled in-vitro (by the same PFC as used for SP) to test for non-invasive in-vivo tracking by 19 F-MRS and 19 F-MRI in mice bearing a B16-OVA tumor. Accordingly, the aim of the study was to develop a reproducible protocol for the in-vitro 19 F-labeling of the three cell groups, to determine the detection limits of 19 F-MRS and 19 F-MRI for the in-vivo detection of these cells, and to test this application in B16-OVA tumor bearing mice.

Animals
All animal procedures were approved by the animal ethics committee (SCAV: Service de la Consommation et des Affaires Vétérinaires, Epalinges, Switzerland). All MR examinations were performed under ketamine-medetomidine anesthesia, and all efforts were made to minimize suffering. Mice were maintained under specific pathogen-free conditions. Ovalbuminspecific TCR transgenic (OT-1) mice were used to produce SP (described below). OT-1 mice were on a RAG1 -/background. CD45.1 C57BL/6 mice were used as recipients for adoptive transfer (described below). Ten days prior to adoptive transfer, tumors were implanted subcutaneously and dorsolaterally with inoculations of 10 6 B16-F10-OVA melanoma cells in 50 μl saline in CD45.1 C57BL/6 mice [21]. The in-vivo protocol is depicted in Fig 1.

T cell isolation and activation
Spleens from CD45.2 + OT1 mice were removed aseptically and homogenized by passing through a cell strainer (40μm). Red blood cells were lysed by the addition of a buffered ammonium chloride solution. The nucleated remaining cells (SP) were resuspended in complete medium (RPMI-1640 medium with 10% FBS, 100 μg/ml each of streptomycin and penicillin, 10 mM HEPES and supplemented with 2-mercaptoethanol) and two different protocols were applied to produce either T act or T OVA-act . The SP population was used immediately after At day 0 (D0) eight CD45.1 C57BL/6 mice received 10 6 B16-F10 melanoma cells by subcutaneous injection in order to induce a malignant melanoma. On the same day SP were prepared from OT-1 mice and two different protocols were applied to generate T act or T OVA-act (as described in Materials and Methods, T cells isolation and activation section). At day 8 (D8), PFC was added in the cell culture medium for 18h in order to label SP, T act and T OVA-act with 19 F. Then, at day 9 (D9) the 19 F-labeled cells were injected IV: 2 control mice (with no tumors) received 50 x 10 6 SP, 3 mice received 20 to 50 x 10 6 T act and 5 mice received 20 to 40 x 10 6 T OVA-act . Finally, 9 mice were imaged at day 10 (D10; 1 T act injected mouse was not imaged) and all mice were immediately sacrificed for subsequent analysis of the organs (liver, lungs, spleen and tumor) by flow cytometry (all mice) and high resolution in-vitro NMR spectroscopy (2 SP injected mice, 3 T OVA-act injected mice and 1 T act injected mouse). The study protocol was performed in a total of 10 animals. In black: in-vivo part; in blue: cell preparation. isolation for in-vitro FITC-conjugated or un-conjugated 19 F-labeling and dead cells were eliminated with Ficoll (GE Healthcare) prior to injection into CD45.1 + C57BL/6 mice. T act were obtained by stimulation of the SP population with recombinant murine IL2 (20 ng/ml), antimouse CD3 (500 ng/ml) and anti-mouse CD28 (0.1 μg/ml) for 2 days. T OVA-act were obtained by stimulation of the SP with the OVA peptide (SIINFEKL; 2 μg/ml) for 2 days in the presence of recombinant murine IL2 (20 ng/ml). In both protocols, two days after stimulation, clusters were formed and harvested to form a single-cell suspension. Dead cells were eliminated with Ficoll and cells were seeded in complete medium containing recombinant human IL15 at 20 ng/ml. Medium was changed every 2 days for one week.

Cell Labeling
SP, T act , and T OVA-act were labeled in-vitro with Cell Sense (CS-1000), a 19 F-based MR imaging agent. Cell Sense is an aqueous colloidal suspension (= nanoemulsion) of a perfluoropolyether perfluorocarbon polymer (PFC), having total fluorine content of 145 mg/mL (Celsense Inc., Pittsburg, PA, USA). The average nanoemulsion droplet size is 180 nm. It is formulated with excipients that facilitate PFC uptake into all cell types, regardless of their ability to phagocytose. The PFC used in Cell Sense is stable at low pH [22]. SP, T act , and T OVA-act were also labeled invitro with FITC conjugated PFC. In all conditions, PFC was added to the cell culture medium at a concentration of 10 mg/mL and incubated with the SP, T act , or T OVA-act for 18 hours at 37°C, 5% CO 2 (SP n = 5; T act n = 14; T OVA-act n = 10). After this incubation period, the cells were washed three times with PBS and counted.

High resolution in-vitro 19 F-NMR spectroscopy of labeled cells
In order to measure the mean 19 F content present in the cells after labeling, quantitative 19 F NMR measurements were performed in lysed cell pellets. A known number of labeled cells (~3x10 6 ) were spun down, resuspended in 250 μl of 1% Triton X100 v/v in PBS to lyse the cells. The cell lysate was mixed with 250 μl of a calibrated 19 F reference solution, trifluoroacetic acid (TFA) at 0.1% v/v in D 2 O, and placed in a 5 mm NMR borosilicate tube. The 19 F NMR measurements were performed using a Bruker AVANCE III HD 400 MHz (9.4 T) NMR spectrometer (Bruker BioSpin AG, Fällanden (ZH), CH). The average 19 F-fluorine content per cell was calculated from the ratio of the integrated areas of the TFA and PFC 19 F spectra, normalized to the total cell number in the lysate. PFC 19 F spectra, acquired with 256 scans and processed with a line broadening of 5 Hz, contain several peaks with a major one located at -93 ppm and the TFA peak at -75 ppm. These two peaks were used for quantitative calculations.

Functional assay of PFC-FITC labeled cells
One hundred thousand SP, T act , and T OVA-act were cocultured with 50x10 5 B16-OVA tumor cells in the presence of anti-CD170a antibody and Golgi Stop reagent (BD Biosciences, San Jose, CA). After 5 hours of incubation at 37°C, cells were washed and stained with fluorescent anti-CD3 and anti-CD8 antibodies at 4°C for 20 minutes. Following fixation and permeabilization, the cells were stained with anti-IFNγantibody and analyzed on a FACS LSRII (BD Biosciences) and BD FACS Diva software.

Cytotoxicity assay of PFC-FITC labeled cells
Fifty thousand SP, T act , and T OVA-act were co-cultured with 25x10 5 B16-OVA tumor cells in the presence of Cytotox red reagent (Essen Bioscience, Ann Arbor, Michigan), according to the manufacturer's instructions. Images were acquired every 2 hours with the Incucyte Zoom System (Essen Bioscience) and analyzed with its software.

Phantom experiments
Different dilutions of TFA (0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M) in 0.3M NaCl were prepared in agarose gel for imaging. 1 H images were acquired with a 9.4T spectrometer (Varian, Palo Alto, CA) using a gradient echo sequence (repetition time (TR) 13.2 ms, echo time (TE) 2.4 ms, signal averages 8, matrix 128×128, field of view 18×18 mm 2 , 3 slices with a slice thickness of 2 mm, total acquisition time 13.5 s). Next, for the 19 F acquisitions at the same locations of the 1 H images, a fast spin echo sequence was used with TR = 500 ms, TE = 3.7 ms; echo-train length 4, signal averages 960 scans, matrix 32x32, field of view 18×18mm 2 , slice thickness 2 mm, and a total acquisition time of 64 minutes.

H and 19 F-MRI
The day after adoptive transfer (24h to 36h post injection, Fig 1) mice were anesthetized with intraperitoneal injection of ketamine: medetomidine (75 mg/kg: 0.1 mg/kg). This anaesthetic combination was chosen to avoid any isoflurane 19 F-MR background signal resulting from its accumulation in the fat pads [23]. The body temperature was monitored with a rectal probe (SA Instruments, Stony Brook, NY) and kept constant at 37.0°C by using tubing with circulating warm water. The animals were placed under a custom-designed 18-mm diameter quadrature surface coil tunable to both the 1 H and 19 F frequencies (400.2 and 376.6 MHz, respectively). To acquire coil-localized spectra of 19 F (128 scans) the coil was positioned at 4 different places, the chest, abdomen, left flank, and the right thigh to cover primarily the liver, the lungs, the spleen, and the tumor, respectively.
In 3 mice, the 19 F spectroscopic signal was deemed sufficient for 19 F-MRI (signal-to-noise ratio (SNR) >200). In these mice a stack of 6 axial 1 H images of the liver was acquired with a gradient echo sequence (repetition time (TR) 29.7 ms, echo time (TE) 1.9 ms, signal averages 4, matrix 128×128, field of view 30×30 mm 2 , slice thickness 2 mm, total acquisition time 15.2 s). Next, a stack of axial 19 F images was acquired at the identical position as the 1 H images using a fast spin echo sequence (TR = 500 ms, TE = 3.7 ms; echo-train length 4, signal averages 960, matrix 16x16, field of view 30×30mm 2 , slice thickness 2 mm, total acquisition time 32 minutes).

Organ collection
Immediately after the MRI session the mice were euthanized by cervical dislocation to harvest the liver, lungs, spleen and tumor. A single-cell suspension was then prepared from the different organs using a cell strainer (70 μm) and RPMI medium. The different cell suspensions were washed once with RPMI and then split into two groups with 5% of the cell suspension used for flow cytometry analyses and 95% for high resolution ex-vivo spectroscopy 19 F-NMR analyses.

High resolution ex-vivo 19 F-NMR spectroscopy of excised organs
The 19 F NMR measurements were performed on the cell suspensions prepared from the different organs described above. Ninety-five percent of the cell suspensions from liver, lungs, spleen and tumor were centrifuged and then resuspended into 250 μl of 1% Triton X100 v/v in PBS to lyse the cells. The cell lysates were then mixed with 250 μl of TFA 0.1% v/v in D 2 O (calibrated 19 F reference solution), and placed in a 5 mm NMR borosilicate tube. The acquisition method used was described previously in the "High resolution in-vitro 19 F-NMR spectroscopy of labeled cells" section.

Statistical analyses
Values are given as means ± standard deviation. Analyses of differences between groups were performed using unpaired Student's t-test and one-way analysis of variance (ANOVA) where appropriate (GraphPad Prism software).

In vitro labeling and function of immune cells
The 3 cell groups were in-vitro labeled or not with FITC conjugated or unconjugated 19 F -PFC in order to assess cell labeling efficiency, and to compare cell viability, phenotype and T cell function. After 18 hours of incubation with PFC the cells were stained by Trypan blue exclusion assay to evaluate the potential cytotoxicity due to labeling. For the 3 cell groups (SP, T act and T OVA-act ), the amount of dead cells after PFC incubation was comparable to the untreated control condition (difference when compared to untreated cells of the same type: 0.5%, 7% and 3% for SP, T act , and T OVA-act , respectively). This result shows that the PFC-based protocol safely labels these cells invitro. Moreover, PFC labeling does not affect the proportion of cell populations, no difference was observed after FITC-conjugated PFC staining (Fig 2). SP are composed of~25% of CD3 + T cells (Fig 2A),~55% of CD19 + non-T cells (Fig 2B),~5% of CD11b + non-T cells (Fig 2C), whereas T act and T OVA-act are composed of only CD3 + T lymphocytes (Fig 2A). In CD3 + T cells, almost all cells are CD8 + T cells (Fig 2A) because they are derived from transgenic OT-1 mice. Fig 3 depicts the 19 F content (i.e. the number of 19 F atoms per cell, for the SP, T act , and T OVA-act cells) after 18h of incubation with the PFC agent quantified by high-resolution exvivo 19 F NMR. The mean 19 F content per cell was similar for the 3 cell groups (Fig 3, overallp = 0.72).
Finally to determine the impact of PFC on T cell function, we performed a series of experiments before and after PFC-labeling. As expected, SP showed a weak response due to the small proportion of CD3 + T lymphocytes (Fig 5). PFC-labeling induced a decrease of response in CD107a upregulation ( Fig 5A) and IFNγ (Fig 5B) secretion assays, but did not impact the cytotoxic capacity of T cells (Fig 5C). Hence, PFC-labeled T cells are able to recognize and kill their target.

Limit of detection of 19 F-PFC-labeled immune cells by 19 F-MRI
In order to determine the limit of detection of the method, a phantom experiment was performed using different TFA dilutions (Fig 6A and 6B). Both 1 H and 19 F images were acquired for each dilution (Fig 6C). Under these conditions the limit of detection for 19 F-MRI was 1.5 x 10 17 19 F spins (at a SNR level of 3), which would correspond to 150'000 cells per voxel of 0.63 mm 3 assuming a cell labeling of 10 12 19 F atoms/cell (Fig 6E) corresponding to 238'000 cells per μl.

In-vivo detection of 19 F-MRI signal
In order to follow the migration of the injected immune cells in-vivo, 19 F-MRS was performed in different anatomic areas (chest, abdomen, left flank and right thigh) of the mice injected   detectable in the right thigh (corresponding to the tumor area) whereas 19 F signal was detectable in the chest, abdomen, and left flank of most of the animals, corresponding primarily to the lungs, the liver, and the spleen, respectively. The 19 F signal measured in the abdomen and in the left flank was significantly higher in the SP injected group compared to the other groups (one way ANOVA, p = 0.0083 and p = 0.0076 respectively, Fig 8). Fig 9 shows representative images of 19 F and 1 H-MRI overlays with a strong 19 F-signal in the liver of 3 animals, i.e. in which the spectroscopic 19 F signal was deemed sufficient for 19 F-MRI (SNR >200). As anesthesia duration was limited, the abdomen was the only area imaged.

Ex-vivo high resolution 19 F-NMR spectroscopy
A post-mortem in-vitro quantitative analysis of 19 F-NMR spectra of different organs (liver, lungs, spleen, tumor) was performed as a reference for 19 F organ content after administration of 19 F labeled cells. The 19 F content of the different homogenized organs is depicted in Fig 8B. 19 F signal was consistently measured in the liver (in 6 out of 7 animals) as well as in the lungs (4 of 7) and occasionally in the spleen (1 of 7). No 19 F signal was observed in the tumors of these animals. Moreover, the 19 F measured signal was higher in SP injected mice compared to T OVA-act injected mice (p<0.0001 in liver and not significant in lungs, ANOVA).

Flow cytometry
In order to confirm the label tracking results from in-vivo 19 F-MRS, in-vivo 19 F-MRI, and exvivo 19 F-NMR, the distribution of the donor T cells in the different organs was determined by flow cytometry according to their expression of CD3, CD8 and CD45.2. There was massive infiltration and proliferation of donor cells in a variety of peripheral tissues, including liver, spleen, and lungs (Fig 10). A small population of these donor cells (CD3 + CD8 + CD45.2 + ) was also found in the tumors, with no significant difference between the 3 cell types. However, contrary to what was observed with in-vivo and ex-vivo 19 F-analyses, the amount of adoptively transferred SP was very low in all the organs analyzed and the amount of adoptively transferred T OVA-act more abundant in the different organs compared to either SP or T act , although not to a significant degree (liver: p = 0.1313; lungs: p = 0.1073; spleen: p = 0.109).

Discussion
In this study 19 F-PFC was used to in-vitro label SP and activated T cells and to follow their migration in-vivo in B16-OVA-melanoma bearing mice using 19 F-MRS and 19 F-MRI.

In-vitro labeling of immune cells and the detection threshold by 19 F-MRI
SP, T act , and T OVA-act were successfully labeled in-vitro, achieving similar 19 F content per cell in the 3 populations ranging from 3 x 10 11 to 1.4 x 10 12 atoms/cell. Labeling of activated T cells is consistent with data published by Srinivas et al. reporting a 19 F loading per cell of 1.7 ± 0.9 × 10 12 19 F/cell [4]. In the present study there are, however, some differences compared to the study of Srinivas. We used a commercially available PFC, while Srinivas et al. used a perfluorinated polyether emulsion prepared in their own lab, which required 3 days of incubation to Highest signals were measured in the chest and the abdomen while no reliable signal was detected in the right thigh. 19 F-MRI of SP and T act injected animals are depicted in Fig 9B (middle and lower panels). label cells, in contrast to the 18h-incubation in the present protocol. Importantly, the labeling procedure used in the current work did not affect cell viability.
With an in-vitro phantom experiment we determined the detection threshold for 19 F-MRI for in-vitro 19 F-PFC-labeled cells. Approximately 150'000 cells with an assumed 19 F loading of 10 12 atoms/cell are detectable in a minimal voxel volume of 0.63mm 3 (measured over 34 minutes at 9.4T at an SNR of 3).
In-vivo and ex-vivo detection of in-vitro 19 F-labeled immune cells by 19

F-MRS and 19 F-MRI
Initially, 19 F-PFC labeled SP were tested in non-tumor bearing mice as a proof of concept. Using in-vivo 19 F-MRS we detected the donor cells in the area of the abdomen, the chest, and the left flank corresponding mainly to the liver, the lungs, and the spleen, respectively (Fig 8A). These in-vivo data were confirmed post-mortem by in-vitro quantitative 19 F-NMR, yielding a similar distribution of 19 F signals as shown in Fig 8B. Then, in a next step, 19 F-labeled T act and T OVA-act immune cells were adoptively transferred into B16-OVA tumor-bearing mice. As with SP cells, donor T act and donor T OVA-act were detected by in-vivo 19 F-MRS in the areas of the liver, lungs, and spleen ( Fig 8A) and these data were confirmed by ex-vivo quantitative 19 F-NMR measurements ( Fig 8B). As for in-vivo 19 F-MRS, the post-mortem 19 F-NMR detected highest 19 F quantities in SP injected animals with most frequent positive findings in livers (6 of 7 animals) followed by lungs (4 of 7 animals) and the spleen (1 of 7 animals). Thus, in-vivo 19 F-MRS allows for cell detection in agreement with the true 19 F distribution in these organs. In addition, the same donor cells were successfully imaged in-vivo by 19 F-MRI in the livers of 3 mice.
Our data show that following IV injection of in-vitro 19 F-labeled cells, the majority of injected cells are trapped in the liver and the lungs (Fig 8B). It has previously been shown that a large fraction of IV-injected CD8 + T cells preferentially migrate into the interstitium of normal lungs [24]. In fact, that study suggested that peripheral homing and retention of CD8 + T cells in the respiratory tract is a mechanism to ensure an adequate number of memory T cells being available at the site of potential future respiratory tract infections.
Using in-vivo 19 F-MRS we detected the donor cells in the area of the abdomen (corresponding mainly to the liver), the chest (corresponding mainly to the lungs and a portion of the liver) and the left flank (corresponding to the spleen and a portion of the liver) (Fig 8B). While MRS is most sensitive for 19 F signal detection, it yields only limited spatial information. However, if sufficient 19 F is brought into the target tissue, MRI is able to detect and image 19 F-labeled SP and T act , as exemplified in Fig 9.

SP and T cell behavior after IV injection
While ex-vivo high-resolution 19 F-NMR was used to determine the true 19 F atom content of the organs, flow cytometry was employed to determine the donor cell distribution in the animals. With this technique, the adoptively transferred cells were identified in the liver, lungs, and the spleen as with 19 F-MRS, but flow cytometry also detected the donor cells in the tumor. As SP with a percentage of 0.06% ± 0.05% of donor cells (CD3 + ; CD8 + ; CD45.2 + ) measured by flow cytometry were detected in the liver by 19 F-MRI, one would expect to also image T act and T OVA-act as they were observed in the organs at a considerably higher percentage than SP (i.e. 0.07% to 0.71%, for T act 0.35% to 2.94% for T OVA-act in the various organs; Fig 10A). However, 19 F was detected by 19 F-MRI in only one T act treated mouse. This low rate of detection could be explained by the fact that following both antibody (against CD3 and CD28) and peptide stimulation, both T act and T OVA-act have the capacity to divide rapidly in-vivo. This proliferation of T cells in-vivo would then induce a subsequent dilution of the PFC content in the daughter cells. This dilution of 19 F signal in the daughter cells can be verified in the present study by comparing the ex-vivo high resolution spectroscopy data to flow cytometry data.
The flow cytometry data also demonstrate that T OVA-act have a better capacity than T act and SP to infiltrate the different organs and the tumor. Both, T act and T OVA-act were produced from OT-1 mice and therefore, both T cells are expected to present predominantly the TCR that recognizes the OVA antigen. Nevertheless, flow cytometry in Fig 10A and 10B demonstrates a trend towards higher infiltration of tumors by T OVA-act . In line with the high presence of T OVA-act in the liver, lungs, spleen, and tumor, they were detected by 19 F-MRS but not at a 19 F signal level that allowed for imaging. This is in contrast to SP, which are detectable by imaging even with their low presence in these organs. The SP represent a heterogeneous population. Besides T and B cells they contain dendritic cells and macrophages, both of which are phagocytic and can accumulate 10 to 1000 times more 19 F compared to T cell populations [25]. Also, as these phagocyte populations are terminally differentiated cells, they do not divide, unlike activated T cells. 19 F-MRI was performed 24h after adoptive cell transfer, A division cycle every 8 hours of the PCF-labeled activated T cells e.g. could already reduce the cellular 19 F content by a factor of 8 at the time of imaging. Taken together, this could explain why despite the relatively high number of T OVA-act and T act detected by flow cytometry in the target organs, a diminished 19 F loading was present in these organs, which limited their detection by 19 F-MRS and 19 F-MRI.
From these data, it might be speculated that non-dividing cells would be best for this type of tracking, such as SP or highly-differentiated T cells e.g. killer cells. Another alternative would be to inject a higher number of 19 F-labeled cells to compensate for dilution according the cell division rate.
Limitations of the study and strategies to improve the detection limit of 19

F-labeled T cells in the tumor tissue
Possible ways to increase the sensitivity of the 19 F-MRI method to detect PFC-labeled T cells in the tumor could be to: 1) increase the 19 F-label content of the injected cells by further optimizing the in-vitro T cell PFC-labeling procedure, 2) enrich the proportion of 19 F-labeled T cells in the injected cell populations (e.g. by sorting PFC-FITC labeled cells by flow cytometry), 3). use preferentially non-dividing T cells to minimize the label dilution effect caused by cell division (which was the most likely reason in our study preventing T cell detection in the tumors), 4) use modified PFCs with shorter T 1 (e.g. by gadolinium-coupling), 5) exploit higher magnetic field strength, 6) develop high performance 19 F coils, and 7) exploit emerging fast pulse sequences. For example, combining this 19 F-MRI technique with compressed sensing could be advantageous with regard to shortening the acquisition time. Compressed sensing was already applied successfully for 19 F-MRI by Zhong and co-workers, but this pulse sequence was not available on our 9.4T system [26].
A potential limitation of the study was the rather rigorous threshold of >200 SNR of the 19 F-spectroscopic signal to proceed to 19 F-MRI. As non-localized spectroscopy yields the entire signal of the volume within the coil, a spectroscopic signal below this threshold does not exclude the possibility for locally high 19 F concentrations that would allow for 19 F-MRI. This notion is supported by the liver 19 F-images after PFC-labeled T act injection, which demonstrate a non-uniform signal distribution in the liver (Fig 9B).

Conclusions
Immune cells, including total SP and activated T cells can be successfully labeled in-vitro by 19 F-PFC and the T cells maintain the capacity to detect and kill the tumor cells after 19 F-labeling. They are detectable after IV administration by in-vivo coil-localized 19 F-MRS in liver, lungs, and spleen. IV-injected SP can also be imaged by in-vivo 19 F-MRI while this is more difficult for T cells. In particular, the proportion and/or 19 F content of the injected 19 F-labeled T cells was too low to allow for tumor imaging. Flow cytometry of liver, lungs, spleen, and tumor demonstrates a higher number of T OVA-act and T act than SP in these organs. The difficulty in reliably detecting 19 F-labeled T OVA-act and T act in flow cytometry-positive organs by 19 F-MRS could be explained by T OVA-act and T act proliferation, which would dilute the 19 F signal in the daughter cells and thus, in the target organs. Non-dividing in-vitro 19 F-labeled cell species appear most promising to be tracked by 19 F-MRS and/or 19 F-MRI.

Author Contributions
Conceptualization: CG HAIY ND JL MI PM LH OM JS.