Cre/lox-assisted non-invasive in vivo tracking of specific cell populations by positron emission tomography

Many pathophysiological processes are associated with proliferation, migration or death of distinct cell populations. Monitoring specific cell types and their progeny in a non-invasive, longitudinal and quantitative manner is still challenging. Here we show a novel cell-tracking system that combines Cre/lox-assisted cell fate mapping with a thymidine kinase (sr39tk) reporter gene for cell detection by positron emission tomography (PET). We generate Rosa26-mT/sr39tk PET reporter mice and induce sr39tk expression in platelets, T lymphocytes or cardiomyocytes. As proof of concept, we demonstrate that our mouse model permits longitudinal PET imaging and quantification of T-cell homing during inflammation and cardiomyocyte viability after myocardial infarction. Moreover, Rosa26-mT/sr39tk mice are useful for whole-body characterization of transgenic Cre mice and to detect previously unknown Cre activity. We anticipate that the Cre-switchable PET reporter mice will be broadly applicable for non-invasive long-term tracking of selected cell populations in vivo.

In the present manuscript the authors presented a novel too l for in vivo tracing of cells combining C re-LoxP system and PET technology using sr39tk reporter gene. They could show the use of this tool for tracing specific group of cells such as platelets, T lymphocytes, and cardiomyocytes. Moreover, they demonstrated the use of this tool for tracing cells in two disease -contexts consisting of inflammation and myocardial ischemia.
This study is of general interest and provided a new tool for in vivo phenotyping although some limitations has to be faced, however this will importantly advance the investigation of cell-fate in developmental and/or diseases related questions.
The manuscript is well writing and the figure well prepared. However, this review feels that some few points have to be addressed before being considered for publication: 1-Minor: Expression of mT before C re recombination (in case of inducible C re) or in non -specific cells are not clearly shown.
2-Minor: Rosa-mT/sr39tk appears in page 1 line 97 and the explanation of "mT" is later in line 108.
3-Page 5: lines 129-133: "We performed in vivo [18F]FHBG-PET imaging studies ( Fig. 2) with C repositive experimental mice that were expected to express sr39tk in the respective target cells (sr39tk+; genotype: C re[tg/+],R26[sr39tk/+]). To evaluate no nspecific tracer uptake, C renegative control animals (sr39tk-; genotype: C re[+/+],R26[sr39tk/+]) were analyzed in parallel." In this paragraph I have 2 comments: Minor: The nomenclature is confusing: C re[+/+] should mean C re -negative? Normally animals will be designated as C repos/+/C reneg/-R26[sr39tk/+] Major: a C re positive R26[+/+] is missing, especially after the data presented in Suppl. 5 where pure WT and C re negative are similar and only the C re positive R26[sr39tk/+] showed an effect in cell numbers in Lymph nodes, Spleen and Thymus. It is well-described that (and depending on the mouse background) C re expression alone can cause some alteration independent of the GOI. Therefore, the sentence in page 7: line 181, "Thus, the reduced number of T cells in C D4/sr39tk mice was apparently mainly caused by expression of sr39tk and not by expression of C re recombinase." Is overestimated. 4-Minor: Page 8: line 233, why 1 mg Tamoxifen for 5 days and why after 4 weeks? 5-General Major: The limitation of this technique in C re -lox-inducible models has to be clearly discussed, especially in proliferative cells. They cannot be traced when they proliferate after induction of the recombination and therefore will dilute the effect of tracing; well-designed protocols have to be providing for it. As in the present manuscript, in the case of myocardial ischemia i.e., it remains the question open whether newly formed Mhy6-expressing cells will dilute the labelling and the reductio n of ([18F]FHBG) uptake and will underestimate the rate of cells viability. 6-Suppl. Figure 3 and 4 Minor: Animals are named differently as stated in the text "mice are denoted "C re promoter/lacZ" mice" and in the figure they are denoted "promoter -C re". In suppl. Figure 3 the WT bgal staining (control) is missing.
Major: A co-staining will be more suitable to claim that the bgal positive cells are of a certain nature (ie. C D31, Mac3). Better magnifications are necessary for stating the staining in th e smooth muscle cells rather than endothelial of bgal. Scale bars are missing.  (2) Line 74 and 75: Explain why it is not possible with current Cre transgenic mice.
We changed the sentence to 'With the currently available R26 Cre reporter mouse lines, however, noninvasive quantitative detection of labeled cells in vivo at the whole-body level is not possible, because detection of the aforementioned reporter proteins relies on either ex vivo methods requiring tissue fixation, invasive methods with a small field of view such as intravital microscopy, or semiquantitative noninvasive methods such as bioluminescence imaging' (line 66-71).
We tried to clarify this statement (now lines 85-92): 'However, transgenic mice with a chromosomally integrated Cre-responsive PET reporter gene have not been described to date. In such a mouse line, Cre-expressing cell populations will be labeled for PET imaging through Cre-mediated activation of reporter gene expression at the genomic level. Once reporter gene expression is activated, cells and their progeny are stably labeled, even if the cells proliferate or change their phenotype, which may lead to a loss of Cre expression. This approach would permit noninvasive long-term visualization of any given cell population for which a respective cell type-specific Cre mouse line is available.'  Figure 3B does not seem to reflect this observation. Please discuss. This is correct. One animal showed relatively increased (but not really 'high') uptake into the untreated ear. We have mentioned and discussed this observation on page 8 (line 220-224): 'Autoradiography detected elevated tracer uptake also in the non-challenged right ears of sr39tk+ mice, in particular in one of the three sr39tk+ animals analyzed (Fig. 3c). This was likely due to scratching and transfer of TNCB from the left to the right ear, thereby, inducing inflammation also in the "non-challenged" right ear.' The PET images shown in Fig. 3b (left) are not from this mouse. For clarification, we prepared the figure below for the reviewer. It shows the individual time courses of [ 18 F]FHBG uptake measured with PET in three individual animals (left; we added the uptake ratio between TNCB-treated and control ear in red) in comparison to the respective ear autoradiographs taken on day 20 (right). Indeed, the mouse with elevated tracer uptake in the non-challenged right ear as revealed by autoradiography on day 20 did also show increased PET signals in the untreated ear (first animal, open circles). This PET signal was similar to the TNCB-treated ear on day 20, but much weaker than the PET signals at the peak of inflammation in the challenged left ears at day 13. In general, changes in [ 18 F]FHBG uptake of the TNCB-treated ears over time were much higher than in untreated ears. Therefore, the comparably minor changes within the untreated ears are visible in the autoradiographs, where animals are shown individually, but not in the original Fig. 3b (right), where the average of all animals was shown.
Based on the reviewer's comment, we have now revised Fig. 3b (right) and show PET signals in individual mice/ears. Along the same line, we have also revised Supplementary Fig. 6 b-d to show PET data in organs of individual animals.

(5) In the discussion the authors, discuss the limitation of tracking T cells in lymph nodes due to background accumulation of tracer in bone and gastrointestinal tract. As far as imaging T cells goes, not being able to track lymph nodes which are major sites of T cell accumulation would be a disadvantage if you want to follow biology.
The authors should therefore also discuss the possibility of extending this approach to using alternate PET probes to sr39TK or using alternate PET reporter gene/probe strategies.
We agree with this notion and have now extended the discussion ( (6) The authors should discuss ectopic activity of CD4-Cre mice in skeletal muscle. Is this unique to CD4 promoter leakiness? Do other promoters also have similar ectopic activity? Please discuss.
We think that the weak (but significant) activity of CD4-Cre mice that was detected in skeletal muscle in the biodistribution analysis (Suppl. Fig. 2e) is not due to activity in skeletal muscle fibers, but likely caused by activity in vascular smooth muscle cells of the blood vessels, which are also the reason for ectopic activity of CD4-Cre mice in the heart. This has been stated/discussed in the manuscript (line 166-168): 'Ectopic activity of the CD4-Cre line in some vascular smooth muscle cells could also explain the weak but significant tracer uptake that was detected ex vivo in skeletal muscle (Supplementary Fig. 2e).'

(7) Discuss pros and cons of Cre approach vs other approaches like Crispr to engineer transgenic mice.
Does the reviewer suggest to include a general discussion on Cre/lox vs CRISPR/Cas technology? If so, we feel this would significantly extend the manuscript, which is already close to the word limit. Also, we are not sure as to how such general discussion would relate to the PET reporter mouse model we describe in this paper. If the reviewer thinks it is absolutely required, please advise us more specifically, and we will add it. Yes, thank you for pointing this out. Changed accordingly.

Reviewer #3:
(1) Minor: Expression of mT before Cre recombination (in case of inducible Cre) or in non-specific cells are not clearly shown.
We have extended Supplementary Fig. 1 (new panel f) with macroscopic fluorescence images of mT fluorescence in various organs of R26-mT/sr39tk mice and added the following sentence to the results section (line 116-119): 'In line with previous publications 18,19 , which used the same targeting vector but different reporter genes, we observed strong and ubiquitous mT expression in organs isolated from R26-mT/sr39tk mice (Supplementary Fig. 1f).' (2) Minor: Rosa-mT/sr39tk appears in page 1 line 97 and the explanation of "mT" is later in line 108.
Thank you for this hint, we have changed the text accordingly: Line 93ff: 'To improve cell tracking in mammals, we generated R26 knock-in mice carrying a transgene for Cre-inducible sr39tk expression under control of the ubiquitous cytomegalovirus early enhancer/chicken β-actin/β-globin (CAG) promoter. Because these mice express membrane-targeted tandem-dimer tomato red fluorescent protein (mT) before Cre recombination and sr39tk after Cre recombination, we named them 'R26-mT/sr39tk' mice.' Line 107: 'Before Cre recombination, mT is expressed from the L2 allele, where "L2" stands for "two loxP sites".'

Minor: The nomenclature is confusing: Cre[+/+] should mean Cre-negative? Normally animals will be designated as Crepos/+/Creneg/-R26[sr39tk/+]
We apologize if the nomenclature we used to describe mouse genotypes caused confusion. We added several clarifications all over the manuscript and hope that with these additions it will be clear to the reader that we are using the '+' symbol to indicate respective wild type alleles: (1) Line 119f to: 'The general appearance and viability of R26-mT/sr39tk Fig. 5b-d).' (4) Legend to Figure 2

Therefore, the sentence in page 7: line 181, "Thus, the reduced number of T cells in CD4/sr39tk mice was apparently mainly caused by expression of sr39tk and not by expression of Cre recombinase." Is overestimated.
We fully agree with the reviewer that Cre expression alone may cause phenotypes that are independent of the expression of Cre-responsive (reporter) genes. Indeed, to test for the effect of Cre expression alone (in the absence of sr39tk), we have analyzed in Supplementary Fig. 5b-e also CD4-Cre transgenic mice that did NOT carry the sr36tk reporter gene. In contrast to CD4-Cre/sr39tk mice (that expressed Cre and sr39tk in T cells), CD4-Cre mice (that only expressed Cre in T cells) did not show major reductions in T cell numbers. These results led us to the conclusion 'Thus, the reduced number of T cells in CD4/sr39tk mice was apparently mainly caused by expression of sr39tk and not by expression of Cre recombinase' (line 185).
To clarify the genotypes used in these experiments and to relativize our observations, we have modified lines 177ff as follows:  Fig. 5b-d). Thus, the reduced number of T cells we observed in CD4/sr39tk mice was apparently mainly caused by expression of sr39tk and not by expression of Cre recombinase.'

(4) Minor: Page 8: line 233, why 1 mg Tamoxifen for 5 days and why after 4 weeks?
Amount and duration of Tamoxifen induction were used according to the publication describing the generation and first use of the Myh6-CreER T2 mouse line (Takefuji, M. et al. 2012Circulation 126(16): 1972-1982. In this publication, loss of myocardial gene expression induced by Myh6-CreER T2 was analyzed 2 weeks after the last Tamoxifen treatment. However, to avoid potential side effects caused by Tamoxifen or vehicle (oil), and to reduce stress to the animals, we performed myocardial infarction 4 weeks after finishing Tamoxifen injection.
(5) General Major: The limitation of this technique in Cre-lox-inducible models has to be clearly discussed, especially in proliferative cells. They cannot be traced when they proliferate after induction of the recombination and therefore will dilute the effect of tracing; well-designed protocols have to be providing for it. As in the present manuscript, in the case of myocardial ischemia i.e., it remains the question open whether newly formed Mhy6-expressing cells will dilute the labelling and the reduction of ([18F]FHBG) uptake and will underestimate the rate of cells viability.
We understand this comment in a way that the reviewer is concerned about the fact that once-labeled cells may lose the reporter gene (or reporter gene expression) when they proliferate. If this is the reviewer's concern, we must respectfully disagree. A central strength of our approach of Cre-mediated activation of reporter gene expression is its stability independent of cell proliferation. The key is that the reporter transgene is stably integrated into the cell's genome and, therefore, inherited to both daughter cells upon mitotic cell division without any "dilution", even over multiple rounds of cell division. As Cre-mediated activation of the reporter gene is based on an irreversible modification of the chromosomal DNA (i.e., excision of the mT-encoding gene cassette), it is very unlikely that cells lose reporter gene expression upon proliferation. The commonly used and well-characterized CAG promoter drives sr39tk expression after Cre-mediated activation of the reporter gene; this promoter is known for strong constitutive/cell-type independent gene expression. Therefore, it is unlikely that reporter gene expression is reduced or lost even if cells change their phenotype upon proliferation. The background behind our strategy for stable cell labeling has been mentioned several times in the manuscript. We also hope that changes in the introduction (lines 85ff; see also comment 3 from Reviewer #1) clarify that cell labeling is not lost upon proliferation of the initially labeled cells.
The reviewer also points out that, in case of myocardial infarction, newly formed cells may dilute the labeled cells so that measurement of [ 18 F]FHBG uptake could lead to an underestimation of myocardial viability. We agree with the reviewer. In the current study, we were not able to discern whether heart tissue was regenerated via proliferation of pre-existing cardiomyocytes (which should have been labeled) or formation of new cardiomyocytes from non-cardiomyocyte progenitor cells (which were presumably not labeled). To clarify potential limitations associated with the use of tamoxifen-inducible CreER T2 in general and in our myocardial infarction study, we added the following text to the discussion ( Minor: Animals are named differently as stated in the text "mice are denoted "Cre promoter/lacZ" mice" and in the figure they are denoted "promoter-Cre". In suppl. Figure 3 the WT bgal staining (control) is missing.
Please, see also point 3 (genotype nomenclature  Fig. 3 and 4).' Major: A co-staining will be more suitable to claim that the bgal positive cells are of a certain nature (ie. CD31, Mac3). Better magnifications are necessary for stating the staining in the smooth muscle cells rather than endothelial of bgal. Scale bars are missing The reviewer suggests to perform double staining of X-Gal positive cells with CD31 or Mac3 to better demonstrate the nature of the positive cells. The reviewer is correct. This would be the ideal way to demonstrate the identity of the positive cells. However, co-staining with the X-Gal stain is rather difficult due to the intensity and color of the X-Gal precipitate. Since CD31 and Mac3 antibodies are specific for endothelial cells and macrophages, respectively, we have revised Supplementary Fig. 4 as suggested by the reviewer, and include higher magnifications as insets into each picture. In these new insets, it is clearly depicted that X-Gal stains the smooth muscle cells of the blood vessels as well as macrophages, whereas CD31 stains the endothelial cells sparing the smooth muscle. In addition, we have included a higher magnification of the macrophages in the lung stained with X-Gal. As also pointed out by the reviewer, we have now added the scale bars in all microphotographs. To allow better evaluation of tracer uptake, we have prepared a new Supplementary Fig. 7 that shows all 'cross-sections' of the hearts in VLA orientation for every PET acquisition for both tracers. Images are in the resolution of the original datasets.