**4.2 Tc-99m methoxyisobutylisonitrile (Tc-99m MIBI)**

Tc-99m MIBI, a lipophilic cationic molecules, was originally developed as a myocardial perfusion imaging agent. Today Tc-99m MIBI is also used for the study of many neoplastic diseases. The mechanism of Tc-99m MIBI accumulation in tumor has been reported to depend on the size of a tumor, the blood flow and the richness of mitochondria in the tumor

Post-Therapeutic I-131 Whole Body Scan in Patients with Differentiated Thyroid Cancer 243

Fig. 13. I-131, Tc-99m MIBI and F-18 FDG images. A 62-year-old woman with metastatic papillary thyroid cancer to the left proximal humerus, clavicle and sternum underwent I-131 therapy with 7.4 GBq (200 mCi). Her serum thyroglobulin level was 260 ng/mL. I-131 RxWBS (A) shows focal hot uptakes in the left clavicle and huemrus. On Tc-99m MIBI WBS, irregular hot uptakes are detected in the sternal area and faint uptake in the left clavicle (B). F-18 FDG PET/CT (C-E) shows multiple hypermetabolic lesions in the sternum, both hilar

Thallium-201 (Tl-201) is a monovalemt cationic radioisotope listed in same group with gallium in the periodic table. The membrane permeability for Tl-201 is almost equal to that for potassium (Elgazzar et al., 1993). It is suggested that intracellular accumulation of thallous ions is due to the transmembrane electropotential gradient (Mullins & Moore, 1960). The primary role of Tl-201 in nuclear medicine is for imaging of myocardial perfusion and viability (Pauwels et al., 1998). Also, Tl-201 chloride has affinity for various malignant tumors, although it is not specific for malignant tumor (Senga et al., 1982). Uptake of Tl-201 in tumor seems to be determined by blood flow, grade of malignancy, viability of tumor cells and tumor necrosis (Pauwels et al., 1998). There are several reports regarding Tl-201 and thyroid cancer. Shiga et al. compared FDG PET with I-131 and Tl-201 scintigraphy and reported that FDG uptake was concordant with I-131 in 38% and with Tl-201 uptake in 94% (Shiga et al., 2001). Senga et al. reported that when tumor showed a positive scan with Tl-

lymph nodes and lungs.

**4.4 Thallium-201**

Fig. 12. Iodine negative and F-18 FDG PET positive case. A 65-year-old female underwent I-123 DxWBS (A) and F-18 FDG PET/CT (B,C) for elevated serum thyroglobulin level (3 to 17 ng/ml) after left modified radical lymph node dissection two years ago. I-123 DxWBS (A) does not show abnormal uptake. On F-18 FDG PET/CT (B), focal uptakes were shown in the left lateral neck (arrow). On axial view of fusion image (C), two hypermetabolic lesions were seen in the left cervical level II. Biopsy revealed recurred papillary carcinoma.

cells (Moretti et al., 2005; Piwnica-Worms et al., 1994; Saggiorato et al., 2009). MIBI irreversibly passes into the mitochondria using the electrical gradient generated by a high negative inner transmembrane mitochondrial potential of malignant cells (Chernoff et al., 1993; Moretti et al., 2005; Piwnica-Worms et al., 1994). Also, there is a report that TSH simulates both F-18 FDG PET and Tc-99m MIBI uptake in poorly differentiated papillary thyroid cancer in vitro experiment (Kim et al., 2009). Even though Tc-99m MIBI is inferior to I-131 scintigraphy in detecting I-131 avid lesions (Al Saleh et al., 2007), it can be applied for patients with elevated serum human thyroglobulin levels but negative I-131 whole body scan (Wu et al., 2003). However, there are several reports that F-18 FDG PET is more sensitive than Tc-99m MIBI SPECT in detecting metastatic cervical lymph node in differentiated thyroid cancer patients with elevated serum thyroglobulin level but negative I-131 whole body scan (Iwata et al., 2004; Wu et al., 2003) (Figure 13).


8. The way forward

are by-products as a result of disruption of normal homeostasis and those involved in signal transduction in response to the stresses [85]. Due to involvement of metabolites in important life processes, the field of metabolic profiling could contribute significantly to the study of stress biology in plants. Both primary and secondary metabolites have been shown to play important roles in responses of plants to drought and salinity stresses. Primary metabolites such as sugars, amino acids and intermediates of Krebs cycle were found with important roles in photosynthetic dysfunction and osmotic readjustment. While, the secondary metabolites such as antioxidant scavengers, coenzymes and regulatory molecules responded to specific stress conditions. Both qualitative and quantitative studies of metabolites in response to abiotic stress are helpful in not only determining the phenotypic response of the plant and screening for stress tolerant lines but also reveal the genetic and biochemical mechanisms underlying the

Drought and salt stresses affect the process of photosynthesis, affecting CO2 diffusion leading to photorespiration and hydrogen peroxide production, causing cell damage [87]. Most recently, Rabara et al. [88] analyzed the metabolomics profile of tobacco and soybean roots and leaves facing dehydration stress. The study revealed highest tissue specific accumulation of 4-hydroxy-2-oxoglutaric acid in tobacco roots and coumestrol in soybean roots; indicating 4-hydroxy-2 oxoglutaric acid and coumestrol can be used as markers for drought stress. Metabolomic analysis of intense drought-stressed grapevine leaves was conducted to reveal induction of several metabolites [89]. Metabolomic profiling of Arabidopsis exposed to drought and heat stresses in combination revealed accumulation of sucrose, maltose and glucose [90]. In tolerant and sensitive thyme facing water stress, metabolomics analysis revealed differential changes in carbohydrates, amino acids, fatty acids and organic acids profiles [91]. Metabolites related to the mechanisms of osmotic adjustment, ROS scavenging, cellular components protection and membrane lipid showed significant changes. Metabolomic and proteomic analysis of xylem sap in maize under drought stress revealed a higher abundance of cationic peroxidases, which with the increase in phenylpropanoids may lead to a reduction in lignin biosynthesis in the xylem vessels and could induce cell wall stiffening [92]. Catola et al. [93] reported that trans-2-hexenal showed a significant increase in water-stressed and recovered leaves respect to the well-watered ones in pomegranate plants. This indicated a possible role of the oxylipin pathway in the response to water stress. Metabolites changes in rice grains during water-stressed and recovery indicated involvement in stress signaling pathways such as gamma-amino butyric acid (GABA) biosynthesis, sucrose metabolism and antioxidant defense [94]. Zhang et al. [95] reported that myo-inositol and proline had striking regulatory profiles in Medicago indicating involvement in drought tolerance. Metabolite profiling of hybrid poplar genotypes revealed that amino acids, the antioxidant phenolic compounds catechin and kaempferol, as well as the osmolytes raffinose and galactinol exhibited increased abundance under drought stress, whereas metabolites involved in photorespiration, redox regulation and carbon fixation showed decreased abundance under drought stress [96]. Concentrations of flavonoids, glycosides of kaempferol, quer-

cetin and cyanidin were found in Arabidopsis during drought stress [97].

Salinity stress has been investigated at metabolite level to reveal the response mechanism. In salinity-stressed barley plants, cell division and root elongation was found associated with accumulation of amino acids, sugars and organic acids [98]. Chen and Hoehenwarter [99] reported that sucrose, fructose, glycolysis intermediates and amino acids levels were altered

stress condition [86].

86 Transgenic Crops - Emerging Trends and Future Perspectives

RNA-Seq and genome sequencing and proteomic techniques/technologies (2D, iTRAQ, MALDI, gel-free, label-free, LC-MS/MS-based technologies) have widened the dimensions of analyzing plant responses to abiotic stresses such as drought and salinity. Recent advances in the omics technologies have contributed considerably to our understanding of the plant abiotic stress-responsive mechanisms. In addition to advancing research in other related areas, emphasis has been on the proteomic analysis specific to whole plants, individual organs, tissues and cells [55]. These technologies are helping to characterize individual proteins specific to different organs, tissues and cells subjected to various abiotic stresses. Advanced proteomic information, coupled with other omics approaches would further strengthen the efforts to develop breeding programs based on identification of novel proteins/genes and their integration through marker-assisted selection. However, further efforts are required to focus on individual target points associated with "Omics" technologies and their application to dissect stress-responsive mechanisms. Research needs to be focused on several fronts such as more studies that target post translational modifications (PTMs), cell type-specific proteome analysis, advanced mapping populations in crop plants and comparative proteomic studies. PTMs of proteins may change their stability, subcellular localization, interactions with other proteins and ultimately proteins functioning. A number of studies revealed the important role of PTMs in protein functioning. Studies have been conducted to analyses protein phosphorylation in maize [108, 109], phosphorylation and ubiquitination in Arabidopsis [110, 111] and glycosylation in soybean [112] under various abiotic stresses. In addition to improved methodologies, identification of more PTMs would unravel functional characterization of important proteins involved in stress-responsive mechanisms and plant adaptation to various abiotic stresses.

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Individual proteins characterization and quantification is essential to fully explore the stressresponsive mechanisms in organs, tissues and cells. However, problems may arise due to the conventional methodologies such as protein detection on 2-DE gels [55]. Improved extraction methodologies may overcome such problems. Poor proteome coverage may be the result while detecting leaf proteome with abundance of RuBisCO that constitutes almost half of the total leaf proteins. However, proteome coverage may be improved with the recently adopted fractionation of crude protein extract. Similarly, quantification of stress responsive low abundance target proteins may be improved through selected reaction monitoring (SRM) technique [113, 114]. Such improved techniques would also help unravel commonly expressed proteins in different organs under multiple abiotic stresses. These advanced techniques coupled with improved bioinformatics approaches may help shed further light on plant responses to abiotic stresses. Recently, transgenic plants conferring abiotic stress tolerance have entered vigorous evaluations under greenhouse and filed conditions. Comparative proteomic studies of these transgenic plants may be helpful to characterize key stress-responsive factors among large number of commonly expressed proteins. Identification of major stress-responsive proteins coupled with advances in transcriptomics, metabolomics and bioinformatics tools would help unravel the complex interactions among stress-responsive signaling pathways. Moreover, omics approaches such as proteomics can be extremely helpful in analyzing post-stress recovery responses in the plants, revealing the key proteins/genes involved in the recovery stage [115].


Measuring the relative number of tumor cells in the tumor lesion before and after treatment, may be useful in response assessment. Highly specific markers are needed. For instance, compounds that target melanin biosynthesis (benzamides) (87) and metallopeptides (88) binding to melanocortin type 1 receptor (labeled MSH analogs) have been used in melanoma, but many other labeled molecules, including antibodies, labeled for SPECT and PET, have shown high imaging performances in terms of sensitivity and specificity (89, 90).

#### Imaging TILs

Monitoring the phenotype and function of tumor infiltrating lymphocytes has long been recognized to be important in adoptive tumor cell therapy (91). This was achieved, in animals as well as in human, by the administration of radiolabeled tracers, usually antibodies or analogs, and SPECT or PET. For example, 64Cu-labeled diabody specific for CD8 was used to assess CD8 T cell density in tumors in mice and treatment related changes (92). Whole antibodies, labeled with zirconium-89 afford similar results (56). Many target antigens have been tested in animal models (56, 58) and CD7 seems so far to be the best candidate to target T lymphocytes, with the lowest toxicity (56).

Surprisingly, in patients, immuno-PET has not been used to detect lymphocytes in tumors, other than through their expression of immune checkpoints, as discussed below. However, labeled IL-2 has been used to visualize lymphocyte infiltrating tumors (77, 93). In a pilot study, patients with metastatic melanoma receiving ipilimumab (IPI) or pembrolizumab (PEMBRO) were subjected to SPECT/CT imaging with 99mTclabeled interleukin-2 in an attempt to detect TILs. In 5 patients (2 treated with IPI and 3 with PEMBRO), metastatic lesions could be visualized with a positive correlation between size and 99mTc-HYNIC-IL2 uptake, both before and after 12 weeks of therapy (93).

Texture analysis and radiomics may also, without administration of tracer, provide molecular information about infiltration of lymphocytes in tumors. In cancer patients, evidence of for the presence of CD3 T cells in tumors have been obtained by MRI texture analysis (94) and for the presence of CD8 T cells by CT radiomics and Artificial Intelligence analysis (95).

#### Macrophages

Macrophages are tissue-resident cells of the innate immune systems that perform a variety of functions in host tissue repair and maintenance of homeostasis. Macrophages are associated with auto-immune and inflammatory diseases and, in oncology, one of the tumor escape factors is the presence of protumor macrophage, tumor-associated macrophages (TAM) that support tumor growth (96). In vivo studies have analyzed the biological role and migration of macrophages using different imaging methods such as fluorescent imaging (97), PET, MRI, and multimodal imaging. Macrophages migration to the inflammatory site after an induction of inflammation was analyzed by in vitro labeling with radioactive iodide-embedded gold nanoparticles (RIe-AuNPs) and PET imaging (76). During inflammatory disease such as arthritis, atherosclerotic plaques, in vivo staining of the macrophage with 111In- or 64Cu-labeled antibodies allowed imaging follow-up, evaluation of therapeutic efficacy and therapy adaption (98, 99). For acute or chronic obstructive pulmonary disease, the recruitment of macrophages was monitored by labeling with amine-modified PEGylated dextran-coated SPIO and MRI (81).

In oncology, macrophages are an important part of the tumor microenvironment and thus a therapeutic target. Indeed, the presence of TAM favors tumor escape. In order to assess their presence in tumors and to analyze the efficacy of therapy, these cells were tracked by immuno-SPECT using 111In-labeled anti F4/80 (100) antibody, by MRI using the contrast agents MPIO (82) and ultra-small iron oxide nanoparticles (USPIO) (101), by BLI in transgenic luciferase mice (84) or by multimodal imaging combining MRI and BLI (85). PET imaging using labeled ligands targeting receptors overexpressed in macrophages, such as the Translocator protein (peripheral benzodiazepine receptor), has also been proposed (80).

#### Imaging Tumor Metabolic Activity

18F-FDG is the most commonly used radiopharmaceutical for imaging tumor metabolism in clinical practice. Its use is based on the increased glycolytic rate in tumors compared to physiologic cells, known as the Warburg effect. However, inflammatory and other metabolically active effector immune cells may contribute to activity uptake in tumor lesions (102). By contrast, lesions with high numbers of proliferative tumor cells are 18F-FDG avid, whereas low 18F-FDG avid lesions have been shown by immunohistology to be infiltrated by activated immune cells. As a result, 18F-FDG is not considered as a marker of immune response and new markers such as amino acids, nucleotides, choline, and receptor ligands have been studied. In hematolymphoid tissues, however, increased levels of deoxycytidine kinase (DCK) expression is found; DCK is the rate-limiting step in the deoxycytidine salvage pathway. The tissue-specific expression of this enzyme allows TABLE 1 |Current tracking cell methods in pre-clinic, depending on the type of labeling (direct, indirect, and transgene) and the modality of imaging (TEP/SPECT, MRI, BLI/fluorescence, and multimodal imaging).

Cell Tracking in Immunotherapy

more specific targeting by, for example, 18F−2-fluoro-D- (arabinofuranosyl)cytosine (18F-FAC), which has been shown to accumulate preferentially in CD8+ T cells and in innate immune cells in mice (103).

18F-labeled 3-fluoro-3-deoxythymidine (18F-FLT) is trapped intracellularly after phosphorylation by thymidine kinase 1 (TK-1) but is not incorporated into DNA since 18F-FLTmonophosphate is a very poor substrate of thymidylate kinase (TMPK). Imaging with 18F-FLT has been evaluated to show proliferation more specifically (18), but effector immune cells that infiltrate tumors are mostly of a differentiated phenotype and do not proliferate. Thus, 18F-FLT uptake in the lymph nodes of vaccinated patients only increased in the presence of antigen-loaded DC, providing the first clinical demonstration that immune responses induced by antigen-specific therapy can be imaged in vivo (102).

In bladder cancer, and presumably in other cancers, correlations have been observed between tumor 18F-FDG uptake and expression of PD-1/PD-L1 (104). Such a correlation may be useful for the selection of appropriate therapeutic strategies.

### Immune Checkpoint Inhibitors: Assessment of Immune Status in Tumor Lesions

Immunotherapy agents do not directly attack tumors but reactivate the immune system by targeting adaptive or innate immunity. Immuno-oncology has been revolutionized by the introduction of immune checkpoint inhibitors (ICI) and the approval of ipilimumab in 2011. ICI are monoclonal antibodies targeting immuno-regulatory molecules on the surface of T cells, antigen-presenting cells, and neoplastic cell populations. Clinical success of reagents blocking the CTLA-4 (cytotoxic T lymphocyte-associated protein 4, CD152) and PD-1/PD-L1 checkpoints (programmed cell death protein 1, CD279; programmed death-ligand 1, CD274) has driven rapid regulatory approval for treatment of patients with both solid and hematologic malignancies (105). Patients treated with immune checkpoint inhibitors (ICI) have objective response rates of 20– 40% for solid tumors, lymphomas, and malignant melanomas. Thus, 60% of patients do not respond to treatment. It may of course be expected that patients with tumors presenting a higher load of tumor infiltrating lymphocytes (TIL) are more likely to respond to anti-PD-1/PD-L1 check point inhibitors (106).

A detailed understanding of the tumor microenvironment, including the identification and quantification of different immune cell subsets, their spatial context, and the expression of these immune checkpoint markers is obviously required to go further with these new therapies (107). Changes in immune cell infiltration and biomarker expression before and after therapeutic intervention are critical parameters for clinical development (108). Thus, assessment of PD-L1 expression by IHC has emerged as an important predictive biomarker for patients with various cancers including non-small cell lung cancer (NSCLC) and renal cell cancer (78).

Immuno-detection using antibodies labeled with zirconium-89 or copper-64 for PET, as well as indium-111 for SPECT, has been used to assess the CTLA-4 and PD-1 status of TIL in vivo and the expression of PD-L1 by tumor cells in order to predict the therapeutic efficacy of the administration of immune checkpoint inhibitors in mice and in human (79, 109–111). This approach was also proposed in the context of anti CTLA-4 therapy (107). Based on tumor biopsies, it appears that some patients with PD-L1-negative tumors show clinical benefit of anti-PD-L1 treatment. Thus, a zirconium-89-labeled anti-PD-L1 antibody (atezolizumab), was used to image 22 cancer patients before atezolizumab therapy. High PET signal was observed in lymphoid tissues and inflammation sites. In tumors, high but heterogeneous and variable across tumors uptake was observed and clinical responses could be better correlated with PET than with immunohistochemistry or other biomarkers (112).

In mice, the presence of CD8+ T-cells was monitored using 89Zr-labeled an anti-CD8 single domain antibody after treatment of B16 melanoma with an anti-CTLA-4, showing that response correlated with the homogeneity of the distribution of CD8+ Tcells through the tumors (58). In mice with B6-F10 syngeneic melanoma, an anti-mouse PD-1 antibody labeled with copper-64 showed tumor uptake (79).

### Monitoring the Activation and Expansion of Immune Effector Cells

Activation and expansion of the immune system may be monitored by imaging changes in the expression of various receptors to cytokines and growth factors as well as changes in the amounts of interstitial water resulting from inflammation. Immune cell trafficking is another aspect of immune system activation.

#### Imaging Immune Cell Activation

Several examples may be found in preclinical and clinical studies. In mice, an antibody against the cytokine IFNγ, which becomes sequestered at the surface of tumor cells after its production by T lymphocytes, was shown to reflect the activation status of cytotoxic T cells (113).

Reactive lymph nodes also express and secrete chemokines that induce immune cells relocation. Among others, the CCR7 chemokine and chemotactic agents, which play a key role in directing cell trafficking, are suitable imaging targets. For example, CXCL12 is a key chemotaxis factor for lymphocytes with expression of the CXCR4 receptor on their cell membrane (114). PET tracers targeting CXCR4 were thus used in cardiovascular disease and infections. Interestingly, Radiolabeled CXCR4 ligands are also very effective for cancer cell imaging (e.g., 68Ga-labeled pentixafor) and CXCR4-trageting therapeutics labeled with 177Lu are currently under clinical development (114).

Activation of the immune system also results in VEGF release and, subsequently, in significant lymph node volume increase. Lymph node volume can be measured using various techniques including MRI, CT, and ultrasound. Ultrasound imaging using targeted microbubbles improves the evaluation of the microvasculature (115). Dynamic contrast-enhanced (DCE)- MRI using gadolinium (Gd) or USPIO-based contrast agents may also be used to monitor angiogenesis: expansion of lymph node size, total blood flow and blood volume, permeability of perfused capillaries, and total surface of perfused capillaries. MRI measures of vascularity using iron-based contrast agents have been validated against histology, the gold standard in angiogenesis assessment. Diffusion-weighted (DW)-MRI detects metastatic lymph nodes (116) and may be able to image reactive LNs in immune responses.

Imaging the expression of VEGF receptor may also be a way to monitor the activation of endothelial cells in LN resulting from immunotherapy. This was achieved in preclinical models by using anti-VEGFR (bevacizumab) labeled with indium-111 for SPECT (117) or by using RGD peptides labeled with various radionuclides for SPECT and PET imaging (118, 119). These approaches have shown potential in mice, for instance, to image inflammation-induced expansion and regression of lymphatic networks by PET, they have not yet been translated into human.

Changes that occur in the tumor due to an increased immune response can also be imaged using MRI, for example through changes in relaxation times, contrast, or apparent diffusion coefficient (120). These changes have been shown to correlate with conventional histological measures in mice after treatment by transferred cytotoxic T cells that expressed a modified TCR specific for a tumor antigen.

#### Imaging Trafficking of Immune Effector Cells

Antigen presenting cells (APC) are cells of the immune system that present pathogen peptides linked to class I or class II major histocompatibility complex (MHC) molecules to T lymphocytes (TL) to initiate adaptive immune responses. They are dendritic cells (DC), macrophages, and B lymphocytes. Analyzing antigen capture, migration to the lymph nodes and antigen presentation by APC started with fluorescently labeled cells using in vivo intravital optical imaging (121, 122). Regardless of the microscope type used, this system remains an invasive process, limited in depth penetration and restricted to a specific area of the body. Thus, APC trafficking has been monitored mostly by MRI and, more recently, tracking methods using PET have been reported. Either the cells or mice are genetically engineered, or labeled antigens are loaded in vivo or ex vivo into APC thanks to their phagocytic capacity. DC may be loaded ex vivo with pathogen peptides or irradiated tumor cells and reinjected to the patient. As an alternative, vaccination using labeled irradiated tumor cells or inactivated pathogens have been used to quantify antigen capture and delivery to lymph nodes by MRI (123).

Imaging lymphocyte trafficking is most easily achieved with ex vivo labeled cells. Transfused cells often traffic initially to the lungs, bone marrow, liver, and spleen. In mice, labeling Th1 cells with 64Cu-PTSM was shown to permit their detection in single LNs and to monitor T-cell homing in vivo over 48 h (117). Changes in cell trafficking resulting from treatments with cyclophosphamide or IL-12 may be monitored by in vivo imaging. A similar method, using zirconium-89, was used to monitor γδ T-cells homing into tumor lesions in mice (75). IL13Rα2-CAR T cells delivered intraventricularly were detectable by PET for at least 6 days throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a nine-fold greater tumor-to-muscle ratio than for CAR-negative T cells. Bone marrow uptake of 89Zrlabeled hematopoietic stem cells could also be monitored in mice (124) and bone marrow cell uptake in acute fractures in mice could be inhibited, rather than accelerated, by a CXCR4 antagonist, plerixafor (125).

The use of reporter gene expression is another way to study cell trafficking, because imaging is independent of factors lifetime and distribution of the tracer and an enzymatic reporter allows for amplification of a weak signal. Antigen-specific T-cells were made to express a viral Tk gene could be tracked in mice, over a period of 3 weeks, using an 18F-tagged probe specific to this variant of Tk. Detection of 104 T cells was claimed (67).

Lymphocytes may also be imaged by targeting cell surface markers. 99mTc-labeled IL-2 was used to detect tumor-infiltrating lymphocytes in melanoma patients (77, 93). Non-depleting 111Inlabeled anti-CD4 antibodies have been used to track CD4+ T cells by SPECT in mice with good correlation with pathologic measures (126). In vivo 19F MRI was also used to track homing to draining lymph nodes of T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion (127). Time-lapse 19F MRI was used to calculate the number of T-cells in lymph nodes over 21 days and correlated with in vitro fluorescence measurements to compensate for in vivo Tcell division. MRI also allowed visualization of CD8+ cytotoxic T cells, regulatory T cells, and myeloid-derived suppressor cells loaded with to monitor the effect of vaccination. Increased recruitment of cytotoxic T cells and decreased recruitment of myeloid-derived suppressor cells and regulatory T cells to the tumor was observed (128).

### Cell-Based Therapies

#### Earlier Results in Cell-Based Therapy

So far, most clinical studies have used 99mTc or 111In or superparamagnetic iron oxide to label therapeutic cells for in vivo cell tracking using SPECT or MRI, as reviewed by Srinivas et al. (129). Adoptive T-cell therapy (ACT) using expanded autologous tumor-infiltrating lymphocytes (TIL) and tumor antigen-specific T cell expanded from peripheral blood are complex but powerful immunotherapies. Clinical trials that included cell tracking have compared various routes of administration, the effect of the number of injected cells or host pretreatment with cyclofosfamide and compared various therapeutic cell preparation and encapsulation methods.

#### Tracking Antigen-Presenting Cells in vivo

DC are the most effective professional antigen presenting cells for the priming of naïve T cells in vitro and in vivo. These properties are the consequence of constitutive expression of MHC molecules class I and II and co-stimulatory molecules (CD80, CD86, CD40) and of their ability to secrete regulatory cytokines such as interleukin 12 upon recognition by the T cell receptor. In the immature stage, DC have the ability to capture the antigen by phagocytosis or endocytosis, migrate to the lymph nodes where they become mature and prime T lymphocytes inducing the adaptive immunity. This is the reason why, in recent years, immunotherapy targeting dendritic cells has developed.

Imaging demonstrated the ability of intradermally or subcutaneously administered therapeutic DC to migrate from the sites of injection into lymph nodes with about 4% of DC reaching draining lymph nodes (130). Actual contact of the DC with Tcells cannot be demonstrated by in vivo imaging, but ex vivo only after lymph node resection. By contrast, intravenously injected mature DC are trapped in the lungs and redistribute to the liver, spleen, and bone marrow. No lymph node localization has been detected so far, which does not mean that DC completely fail to reach the lymph nodes. The techniques may not be sufficiently sensitive to detect the small numbers of cells that do reach the lymph nodes. Direct intranodal administration of therapeutic DC is also common in clinical studies. Then in vivo imaging has been used to study the migration of DC from the primary injected node to secondary nodes. The large variability in the fraction of injected cells (from 0 to 84 %) that was shown to migrate cast doubts on the accuracy of intranodal injections. Labeling the antigen to monitor its fate after DC delivery has been proposed in preclinical in vivo models. When DTPA was conjugated to the epsilon NH2 group of the Lys154 residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allowed the non-invasive determination of the behavior of MHC–peptide complexes expressed by DC in cell vaccination (131) but has not yet been reproduced in the clinic.

#### CAR (Chimeric Antigen Receptor) T-Cell Therapy

CAR T-cell therapy is a fast-growing research field with various therapeutic indications in autoimmunity, allotransplantation, infection and cancer. Enhancing the functionality and the safety of the injected cells is an important aspect of the clinical development of this very potent therapy. Therefore, there is a real need to develop in vivo molecular imaging to better visualize, predict and improve the efficiency of this type of immunotherapy (5). However, so far, clinical studies of CAR T-cell tracking have only established proofs of concept of its feasibility (74).

SPECT and PET imaging are two possible modalities for tracking the fate of T-cells injected for therapeutic use. Labeling T-cells has been extensively investigated and radiolabeling is possible with little impact on cell function or migration ability (13). However, the radionuclide half-life is a limitation to track the cells more than for a few days after injection.

A variety of solutions to this limitation have been proposed (132). While multimodal imaging has been shown possible (133), CAR T-cell tracking in animals has demonstrated homing and persistence in the tumors and spleen by ex vivo MRI of tissue samples after CAR T-cell labeling with perfluorocarbon (134) and in whole animals by immuno-PET (135). Although the context of CAR T-cell therapy would be appropriate to develop genetic modification of the T-cells to express reporter genes as discussed above, the outcome of therapy remains monitored mostly by functional imaging and especially by MRI (136).

#### Other Adoptive T-Cell Transfer Therapies

Although BLI is most of the time used to follow tumor and stem cells, T cells could also be monitored thanks to the luciferase gene; for instance, the migration of CD8 T cells toward tumor site was evaluated in a xenograft mouse model (83), and the migration of tumor associated macrophages has been visualized in a transgenic mouse model of ovarian cancer (84). Also, by optical imaging, but using a fluorophore targeted to NIS-transfected cells, tracking of ex vivo-expanded NK cells has been performed in vitro and in vivo showing fast NK cell accumulation in tumors in triplenegative in breast cancer xenografts (137).

89Zr-oxinate labeling was used to track Vγ9Vδ2 T cells in vivo by PET. In a mouse xenograft model of human breast cancer, the Vγ9Vδ2 T cells could be tracked over 1 week and it was shown that injection of PEGylated liposomal alendronate increased homing of the T cells to the tumors, which was confirmed by histology (75).

#### Stem Cell Therapies

Mesenchymal stem cells (MSC) have been proposed for cardiac regeneration after myocardial infarction (MI). Mesenchymal stem cells derived from rat fetal heart have the potential to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. These cells were labeled with technetium-99m for in vivo tracking that revealed a focal uptake of cells in the anterior mid-ventricular region of the heart in line with subsequent ventricular functional recovery (138). Cardiac stem cells were also loaded with 18F-FDG and imaged by PET to quantify their biodistribution and assess the retention of implanted cells in a model of chronic myocardial infarction in pigs. Acute cell retention was shown not to correlate with cell engraftment, which is improved by IM injection (17).

Stem cells have been tracked in various models with BLI, for instance in acute liver injury or acute kidney injury, to study the migration and persistence of human bone-marrow derived stem cells to the liver and kidney (45, 139). Luciferase-transfected adipose-derived stem cells could be transplanted in liver and brain and monitored in vivo by bioluminescence for several days. Ex vivo, immunofluorescence detected the continued expression of luciferase for 4 months, demonstrating that the transplanted cells do not dye, even if the bioluminescence signal is lost (46).

In a tumor graft model, the migration of mesenchymal stem cells toward the subcutaneous tumor could also be observed (140). This study took advantage of a different type of luciferase that metabolizes different substrate, allowing them to follow the migration of stem cells with the Firefly Luciferase, and the tumor progression with the Renilla Luciferase.

BLI imaging has allowed to investigate the impact of stem cells injection modalities, showing that the intravenous route often leads to sequestration in the lung (141) preventing the migration of stem cells to other organs, while the intracardiac route seems to prevent this phenomenon.

#### DISCUSSION

Cell tracking has a long history of routine clinical use in Nuclear Medicine and it serves a purpose in infectious and inflammatory diseases despite its limitations (142). Imaging has an increasing role in the context of personalized medicine, which becomes the approach to take, at least in developed countries. CT-scan, MRI and ultrasonography are now inescapable and Nuclear Medicine modalities have gained larger recognition, particularly in cancer. However, the number of tracers of frequent, routine use remains quite limited. In addition to bone and thyroid scans, 18F-FDG is certainly the tracer that has the biggest impact in cancer management, with a few other PET tracers for those cancers in which 18F-FDG does not perform so well, such as prostate cancer. In view of the incredible number of preclinical and early clinical studies about cancer imaging, this seems not much. There are many obvious reasons, the major one being the difficulty of demonstrating that a new imaging technique has its place in medicine as compared to all existing ones. If the imaging technique needs an injectable tracer, such as a radiopharmaceutical, the situation is worse, because of the cost of developing a product that has the regulatory status of a drug and by far not the sales and price of a therapeutic compound.

Will immunotherapy change this situation? Most of the very large number of original publications and reviews that deal with immunotherapy advocate for more imaging, especially more specific imaging of receptors, antigens and other biomarkers that characterize the function of cells in vivo. With the progress of cellular therapies, whether regenerative or cancer-oriented, many papers call for cell tracking in vivo as a way to understand their behavior and mechanisms of action and, by the way, to design improved therapies. Indeed, if not all novel immunotherapies are cell therapies, they all bear upon complex cellular interactions at the tumor sites and in immunologic tissues and better knowledge of the nature of cancer and non-cancer cells residing in tumors, their activation, proliferation and migration in living animals and, of course, in humans, must be a way of progress in therapy.

This realization triggered a lot of developments that made possible better cell labeling, mostly to make them visible by MRI and PET, for longer times after re-injection, as well as improved tracers to target specific biomarkers in vivo, using SPECT and PET, but also MRI, not only on tumor cells but on those cells that make the tumor microenvironment, e.g., endothelial cells, infiltrating antigen-presenting cells, lymphocytes, macrophages and other cells of the immune system. New techniques have been developed and the use of reporter genes to make cells detectable any time after their inoculation using specific tracers is a particularly elegant and powerful one. This review has rapidly depicted these approaches and it is expected that it convinces the reader that they are feasible and effective.

There is no best technique, though. It depends on the objective and, obviously, the most powerful one, for instance the use of reporter genes, are associated with complex manipulations, cost and regulatory hurdles. Interestingly, radioactive (SPECT, PET) and non-radioactive (MRI, optical imaging, ultrasonography) methods have been proposed, which all have advantages and drawbacks. Of course, bimodal and even multimodal agents have been developed. Multimodal imaging is clearly the way to go, with SPECT and PET now always associated with CT and PET-MRI systems developing. It is also clear that multimodal imaging experiments in animals that allowed for in vivo imaging and ex vivo in-depth investigation of the fate of injected cells and confirmation of in vivo imaging results are most convincing. However, such studies do not necessarily need bifunctional tracers, which sometimes look more like "tours de force" than candidates for further development.

One question here is: have these developments and studies been useful for the development of immunotherapy? The answer is not obvious. Such studies have pointed out to some problems and they have been mostly confirmatory, when they have not merely been proofs of concept for feasibility. This review has attempted at presenting clinical results in cell tracking and, while it may have missed some, it is in line with other recent reviews on the subject to conclude that the number of clinical studies is quite small. It may be considered that this is only the beginning of a new story and that groundbreaking discoveries in immunotherapy will be made thanks to imaging and that at least some of the approaches reported here will find their application. Very few cell tracking techniques will become routine. The introduction of reporter genes in therapeutic cells is probably the technique with the highest sensitivity for long term monitoring of cell trafficking, proliferation, and persistence. For cell therapies in which the cells are genetically modified, namely CAR-T cells, the addition of a second gene and cell tracking may be considered in the context of clinical trials. Whether this approach will be used in routine clinical practice is not likely. Conversely, tracers for in vivo imaging, particularly PET imaging, designed to detect and quantify specific cell populations are being developed and some will find a routine use. It is always difficult to make predictions, but it seems logical that expensive therapies or therapies that may be efficacious but associated with serious side-effects will not be given to patients who have no chance to benefit from them. The imaging of tumor microenvironment may give answers to how the patients will respond to such therapies, especially immunotherapy. Expression of immune checkpoints, like anti-PD-1, is already assessed from biopsies prior to immunotherapy, but the use of PET-imaging or MRI could allow a non-invasive assessment of the immune state of the tumor. This could provide new insights into the prediction of the response to treatment in patients. This is the theragnostic approach, which is not a reality today, but most probably be one in the future. It is also quite probable that future research in immunotherapy will take advantage of all these technological advances, certainly for preclinical studies, but also in the clinic. Indeed, it is time to combine the novel therapeutic approaches, which afford impressive remissions but not yet to all patients and this will call for precise, specific understanding of what is really going on in the living organism.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

This work was supported in part by grants from the French National Agency for Research called Investissements d'Avenir IRON Labex no ANR-11-LABX-0018-01 and ArronaxPlus Equipex no ANR-11-EQPX-0004.

#### REFERENCES

dopamine type 2 receptor and 18F-fallypride. J Nucl Med. (2014) 55:1342–7. doi: 10.2967/jnumed.113.134775

tumors activated with liposomal alendronate. Mol Ther. (2019) 27:219–29. doi: 10.1016/j.ymthe.2018.10.006

checkpoint inhibitor therapy: a pilot study. J Clin Oncol. (2017) 35:3034. doi: 10.1200/JCO.2017.35.15\_suppl.3034

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Perrin, Capitao, Mougin-Degraef, Guérard, Faivre-Chauvet, Rbah-Vidal, Gaschet, Guilloux, Kraeber-Bodéré, Chérel and Barbet. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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**2. JNK-SAB-ROS Activation Loop**

Interest in the role of JNK signaling in liver injury began nearly 20 years ago with the identification of the protection of cultured mouse hepatocytes from acetaminophen (APAP)-induced necrosis by a JNK inhibitor (SP600125) [5]. This was further supported by the discovery of protective effect in in vivo JNK1 and 2 double knockdown using antisense oligonucleotides [6]. In this work and later in other in vivo and in vitro models of JNK-dependent liver apoptosis (e.g., TNF/D-galactosamine [TNF/GalN], tunicamycin-induced endoplasmic reticulum (ER) stress, and lipoapoptosis), a key important finding, i.e., the association of P-JNK with mitochondria, was discovered. Then, the binding target of P-JNK on mitochondria and the significance of this interaction were uncovered [7–9]. This seemed particularly relevant as earlier works had suggested that mitochondrial ROS played an important role in the sustained activation of JNK and that antioxidants could protect against sustained JNK activation and apoptosis in response to TNF [8,10].

One prior publication by Wiltshire et al. had identified a mitochondrial outer membrane protein—SAB (SH3BP5)—as a target of P-JNK binding and substrate for JNK phosphorylation [11]. These studies were performed in chicken embryotic fibroblast cells, and the functional consequences of the interaction were not further explored [11]. Then, SAB was identified exclusively in the outer membrane of mitochondria in liver [12]. SAB and P-JNK coimmunoprecipitated shortly after toxic stress from APAP prior to overt liver injury [7,13]. The finding was further supported by adenoviral sh-SAB (versus sh-lacZ control)-mediated depletion of SAB in liver. Knockdown of SAB resulted in inhibition of sustained JNK activation and translocation to mitochondria in all the models of JNK-dependent toxicity (APAP, TNF/GalN, ER stress, palmitic acid lipotoxicity) in vivo or in vitro. This was accompanied by striking protection against cell death [7–9]. More recently, these findings have been fully confirmed using hepatocyte-specific inducible knockout of SAB in two-month-old mice either after crossing *SABfl/fl* mice with transgenic tamoxifen inducible *alb-CRE+/*<sup>−</sup> mice followed by tamoxifen feeding or by injection of *SABfl/fl* mice with hepatocyte-targeted CRE viral vectors (adeno-alb-CRE or AAV8-TBG-CRE) [12].

Another important issue to address is the effect of the interaction of P-JNK with SAB on mitochondrial function and ROS production. Using isolated normal liver mitochondria, recombinant P-JNK1 and/or 2 in the presence of ATP was shown to lead to inhibition of oxidative phosphorylation and maximum respiratory capacity [12]. This effect was not observed in the absence of ATP, suggesting that phosphorylation of SAB was required. Furthermore, this effect was accompanied by enhanced O2 production in MitoSOX-loaded mitochondria [8,14]. The effect of P-JNK + ATP was absent in liver mitochondria from SAB knockout mice and was inhibited by a peptide corresponding to the JNK docking site of SAB, which blocked the interaction of JNK and SAB [8,12].

The topology of SAB can be defined using C- and N-terminal-targeted antisera. The short C-terminus faces the cytoplasm and contains a JNK kinase interaction motif (KIM), which is the docking site. The longer N-terminus faces the intermembrane space [12]. Since there is no evidence that JNK enters the mitochondria, the question relates to how the interaction of P-JNK with SAB and its phosphorylation on the external face lead to impairment of mitochondrial bioenergetics. The mechanism of JNK-SAB-mediated impairment of mitochondrial respiration has been explored [12]. Tyrosine-protein kinase c-SRC, mainly in the P-419-SRC active state, has been shown inside mitochondria of liver and neurons and is required to maintain the function of the electron transport chain. SRC kinase inhibitors reproduce the same effect as P-JNK/ATP on isolated mitochondria. When mitochondria were exposed to P-JNK/ATP, rapid dephosphorylation of P-SRC was observed, but this did not occur in mitochondria from SAB knockout liver and was also inhibited by the KIM blocking peptide [8,9,12]. Furthermore, inactivation (dephosphorylation) of SRC occurred in liver mitochondria after in vivo treatment with APAP or TNF/GalN. Inactivation of SRC was inhibited in isolated mitochondria after treatment with vanadate, which blocked the effect of P-JNK/ATP on mitochondrial respiration. The study indicated that a phosphotyrosine phosphatase (PTP) was responsible for mediating the effect and that intramitochondrial protein tyrosine phosphatase non-receptor type 6 (PTPN6/SHP1) was responsible for inactivation of SRC when JNK interacted with SAB on mitochondrial outer membrane. Mitochondria isolated from PTPN6-depleted mice were resistance to the effects of P-JNK/ATP on P-SRC and mitochondria respiration [12].

Mitochondrial SRC associates with docking protein 4 (DOK4), a kinase and PTP docking protein [15], and DOK4 participates in the JNK activation of intramitochondrial signaling pathway [12]. DOK4 is found exclusively in the mitochondria fraction and is associated with the inner membrane but accessible to the intermembrane space. Knockdown of DOK4 in vivo protected isolated mitochondria

from the effect of P-JNK/ATP and protected against liver injury and sustained JNK activation. The effect of DOK4 knockdown was analogous to the knockout of upstream SAB or knockdown of PTPN6. Careful mitochondrial subfractionation and immunoprecipitation studies have revealed that under basal conditions, SAB is in the outer membrane and bound SHP1 (on the intermembrane face), while DOK4 and P-SRC are on the inner membrane. Following toxic stress and JNK activation, PTPN6 coimmunoprecipitation with SAB decreases in the outer membrane fraction and binding to SRC/DOK4 (coimmunoprecipitation) increases on the inner membrane. Thus, when SAB is phosphorylated by P-JNK/ATP on the cytoplasmic face, PTPN6 is released and interacts with P-SRC, dephosphorylating (inactivating) SRC in a DOK4-dependent fashion. Thus, DOK4 appears to serve as a platform, which is required for the interaction of PTPN6 and P-SRC. PTPN6 associated with SAB is inactive (nonphosphorylated); however, when associated with P-SRC, it is phosphoactivated by SRC, which then leads to dephosphorylation of SRC. Inactivation of SRC leads to impaired mitochondrial respiration and increased ROS release from mitochondria. ROS then activates ASK1, and possibly MLK2/3, which sustains activation of MKK4/7, leading to sustained JNK activation (Figure 1). ROS oxidize thioredoxin, relieving ASK1 of inhibition of dimerization and allowing self-activation of ASK1. ROS also activate SRC at or near the plasma membrane, which then activates MLK2/3. These MAP3 kinases then activate MAP2 kinases, which activate JNK.

**Figure 1.** P-JNK-SAB-mitochondria-ROS-mediated JNK activation loop. JNK activation is triggered by physical and chemical stress, including alterations in nutrients, growth factors, cytokines, extracellular matrix, DNA damage, drugs, and toxins. Activated JNK translocates to mitochondria and interacts with SAB, leading to a sequence of events, i.e., inhibition of intramitochondrial c-SRC activity and mitochondrial electron transport chain and thus release of ROS, which further activates ASK1, MKK4/7, and JNK. P-JNK-SAB-ROS activation loop drives sustained JNK activation, and cell death occurs. Black arrows indicate activation. Blue circulating arrows indicate vicious cycle. Red "T" arrow indicates inhibition of electron transport chain (ETC).

The duration and degree of sustained JNK activation mediates many consequences, both through transcriptional regulation by AP-1 targets that modulate expression of many genes involved in proliferation as well as inflammation (cytokines and chemokines), metabolic gene dysregulation e.g., gene repressors, such as nuclear receptor corepressor 1 (NCOR1) action on peroxisome proliferator activated receptor alpha (PPARα) and thioredoxin-disufide reductase (TR), or through direct activation of proapoptotic BH3 family members and inhibition of antiapoptotic Bcl2 family members (see Reference [3] for review).

#### **3. Modulation of JNK Activation Loop**

MAP kinase cascade, which senses cellular and extracellular stress, conveys cellular response to regulate cell fate. The timing and duration of JNK activation determine whether cells proliferate or adapt to metabolic or toxic stress or undergo programmed cell death, such as apoptosis, necrosis, and possibly other forms of cell death. Thus, modulators of the JNK-SAB-ROS activation loop (Figure 2) and molecular structure of components in the loop (Figure 3) determine the duration of JNK activation and selectivity and specificity of JNK-mediated cellular responses. As the expression of isoforms of kinases, phosphatases, substrates, inhibitors, and scaffold proteins involved in the JNK activation loop are tissue and cell-type-specific, we will discuss the general principles of modulation of the loop.

**Figure 2.** Modulation of JNK activation loop. JNK-SAB-ROS activation loop can be modulated at all level of MAP kinase cascade through phosphorylation by upstream kinase, dephosphorylation by phosphatase, acetylation by sirtuins, deacetylation by p300, protein stabilization by deubiquitinating enzymes, and protein degradation by ubiquitinating enzymes.

**Figure 3.** Schematic diagram of molecules involved in JNK activation loop. JNK is activated by dual threonine–tyrosine phosphorylation at (TXY) located within activation loop (indicated as black dotted line) by MKK4/7 through interaction with common docking site (CD) of JNK and docking motif (D-motif; JNK binding site indicated by yellow bars) of MKK4/7. CD of JNK is also shared with JIP and SAB which has one hydrophobic transmembrane spanning domain (indicated as dotted line). Two JNK binding sites on SAB are noted as KIM1 and KIM2 (indicated by yellow bars). Dimerization of ASK1 through interaction of N-terminal coil-coil domains (N-cc) and of C-terminal coil-coil (C-cc) doamins autoactivates ASK1. Removal of thioredoxin (indicated by gray polygon) by oxidation facilitates dimerization of ASK1.

#### *3.1. MAP Kinases—JNK and p38*

JNK and p38 are activated by MKK4/7 via dual phosphorylation of a Thr-Pro-Tyr (TPY) motif of the activation loop (A-loop), which connects N-terminal and C-terminal lobes. The ATP binding site lies between the lobes [16]. One critical feature of JNK signaling is the use of its single common docking site (CD) to interact with JNK-binding domain (D-motif) of upstream MKKs, MAP kinase phosphatases, substrates, inhibitors, and scaffold proteins. Thus, JNK activity derives from the ensemble of interactomes that compete for the docking site on JNK. Therefore, reducing the level of a single substrate of MAPK can lead to decreased amounts of active MAPK due to loss of competition and greater access of phosphatases to allow graded response. The specificity of JNK signaling relies on hydrophobic φ-x-φ docking motif (D-motif) on JNK substrates and scaffold proteins, such as c-Jun, ATF2, JIP, and SAB, and their subcellular localization. Preferences for D-motifs on different JNK substrates have been analyzed using 11-mer peptides derived from different substrates [17]. SAB peptide derived from KIM1 D-motif is similar to JIP1 sequence but has 21 folds lower affinity for P-JNK. This appears to be due to adjacent Pro residue. This means that small changes in D-motif and adjacent residues can profoundly impact JNK signaling. Indeed, JIP1 has higher affinity for JNK3 than ATF2 or SAB. Cytosolic JIP is a platform to bring together upstream kinases MKKs, MLKs, and Rac for JNK activation [18–20]. The depletion of JIP in MEF cells prevents fatty-acid-induced JNK activation [20]. JNK phosphorylates c-JUN on Ser63/73 and increases c-JUN-dependent transcription and cell proliferation [1], whereas JNK interaction with less affinity to SAB localized on mitochondria accounts for ROS generation, sustained MAP3K to JNK activation, and cell death [7–9,12,21]. Importantly, JNK phosphorylates p53 on Thr81 and stabilizes and confers its transcriptional activity [22], which could dampen expression of SAB and the JNK activation loop (see below).

A distinct difference of p38 compared to JNK is the activation of downstream MAPK-activated protein kinase (MAPKAPKs), such as MK2/3, MNK1/2 [23]. p38 regulates MK2-mediated TNF-α and IL-6 production by promoting translation and/or stability of their mRNAs [24]. p38 modulation upregulates antioxidant response via NF-κB [25] and interferes with ROS produced by the JNK activation loop. p38-mediated MK2 activation also phosphorylates MDM2 on Ser157 and Ser166, resulting in MDM2 activation and degradation of p53 [26]. p53 expression and activity, contributed to by multiple signaling pathways [27–29], importantly regulates SAB expression and determines susceptibility to promoting the JNK activation loop (abstract, manuscript in preparation). The depletion of p53 or inhibition of p53 by pifithrin leads to higher P-JNK levels and more severe necrosis in acetaminophen-induced acute liver injury [30]. Recently, we uncovered an important regulatory role of p53 in SAB expression and thus the contribution of functional p53 in decreasing JNK-mediated cell death (see below). In addition, human rhabdomyosarcoma cells lacking functional p53 undergo rapid apoptotic cell death in mild cellular stress conditions, such as serum-free condition and inhibition of mTOR, through the ASK1-JNK activation-mediated pathway [31]. The mechanism may be by the upregulation of SAB expression in p53 deficiency, which we will discuss further below. This could be a potential therapeutic pathway to target treatment of functional p53-defective tumor cells.

Dual-specificity protein phosphatases (DUSP), which are also known as mitogen-activated protein kinase phosphatases (MKP), are a family of threonine-tyrosine dual-specificity phosphatases that dephosphorylate and inactivate MAPKs such as extracellular regulated MAP kinase (ERK), p38, and JNK in a context-dependent manner [1,32]. The highest levels of DUSP1 are observed in the heart, lungs, and liver. *DUSP1* or *DUSP5* KO mice were shown to exhibit increased JNK activity due to physiological levels of reactive oxygen species, supporting the role of phosphatase in the JNK activation loop [2,10]. Indeed, DUSP inhibition may be sufficient to induce prolonged activation of JNK following some stimuli. DUSPs localize in cytoplasm and nucleus, but mitochondrial localization or association has not been reported. Indeed, DUSP1-mediated dephosphorylation of JNK is sufficient to inhibit JNK-SAB-ROS activation loop because activation of JNK is required for translocation and interaction with mitochondrial SAB. In breast cancer, an inhibitor of DUSP1 is considered an adjuvant

and/or neoadjuvant therapy by enhancing cell apoptosis. The involvement of p38 in modulation of JNK activation loop is more complex because p38/ERK upregulates DUSP1 expression through MAPK-activated protein kinase MSK [23].

In addition to phosphorylation and dephosphorylation, direct regulation of JNK activity also involves participation of acetylation/deacetylation. Cytosolic and nuclear shuttling SIRT2 deacetylates and activates JNK, whereas JNK is inhibited by p300-mediated acetylation. In addition, SIRT2 deacetylates and inhibits DUSP1 [33]. Thus, sirtuin activation occurring in excess nicotinamide adenine dinucleotide (NAD+), such as in nutrient excess or granulocyte-colony stimulating factor (G-CSF) signal transduction, changes the balance of JNK activation. However, JNK activation by sirtuin inducers [34] cannot reach the threshold level to cause sustained JNK activation and therefore does not favor cell death. In addition, sirtuin 1 (*SIRT1*) knockout mice were shown to exhibit less severe liver injury through preconditioned enhanced NF-κB response and dampening sustained JNK activation in the GalN/LPS-induced apoptotic cell death. However, acetaminophen-induced liver injury, which overrides NF-κB-mediated upregulation of antioxidant genes, is not protected [35]. Overall, the contribution of sirtuins in cell death pathways appears to be of minor importance. So far, the role of SIRT3, 4, and 5 in mitochondria have not been explored in the mechanism of JNK-SAB-ROS activation loop [36]. In summary, p38, p53, DUSP1, SIRT2, p300, NF-κB, Gadd45β, and SAB all have the potential to directly or indirectly modulate JNK activity in a complex, context-dependent fashion.

#### *3.2. MKKs—MKK4 and MKK7*

MKK4/7 interacts with JNK via D-motif and phosphorylates and activates JNK on JIP platform [37]. In MEF cells, MKK4 also activates p38. MKK7 contains three JNK docking D-motifs within its 100-amino acid regulatory domain. The second docking site of MKK7 binds to JNK via two alternative binding modes [38]. However, the significance and selectivity of the D-motif of MKK7 on JNK needs further exploration. Interestingly, Ser403 of MKK7 in HEK293 cells is phosphoactivated by serine/threonine-protein kinase ULK1/2 (ATG1/2), which is a downstream target of AKT/mTOR signaling pathway. The significance of this crosstalk in the sustained JNK activation loop in disease models requires further exploration as liver-specific deficiency of ULK1/2 in KO mice were shown to delay and partially protect liver injury from acetaminophen-induced hepatoxicity but not GalN/TNF liver injury [39]. Signalosomes, such as receptor complex in TNF-induced JNK activation and TRAFII-mediated JNK activation in ER stress, are important in integration of crosstalk, induction, amplification, and inhibition of the JNK activation loop [40]. The cytosolic JNK interacting protein (JIP) platform is crucial for initial activation of JNK in fatty acid but not in TNF-induced JNK activation [20]. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV which is required for activation of Rac and downstream activation of MLKs, MKK7 and JNK activation on JIP1 platform in MEF cells, white adipose tissue, and muscle, but other isoforms of JIP could be essential in liver.

The complexity of JNK activation loop is also illustrated by retroinhibition of MAPK cascade by p38a in receptor-mediated cell death [41]. Indeed, p38a is activated by MKK4 and MKK3/6, and p38 then contributes inhibitory crosstalk to the JNK activation loop via inhibitory phosphorylation of TGF-beta activated kinase 1 (TAK1/TAB1), MKK3/6 and/or increased expression of DUSP (MKP) by p38a/MK2 pathway [42–44]. However, this regulatory pathway requires further exploration. In fact, p38 affects several alternative pathways to interfere with the JNK activation loop, such as NF-κB-mediated upregulation of antioxidant genes and 17 kDa Gadd45β (MyD118) [45,46]. Endogenous Gadd45β and MKK7 associate through direct, high-affinity contact. Gadd45β, which is not a phosphatase, inhibits MKK7 by masking the kinase domain. The association is tighter than JIP1 and thus spatially prevents MKK7 activation of JNK. Gadd45β does not inhibit MKK4, MKK3b, or ASK1 activation or phosphorylation of MKK7. Gadd45β expression is suppressed by orphan nuclear receptor small heterodimer partner (SHP), which is a transcriptional corepressor. Depletion of SHP increases Gadd45β expression and prevents sustained JNK activation and liver injury. Additionally,

activation of Akt in a parallel survival pathway could activate NF-κB and inhibit MAP3K, such as MLK3 and ASK1 [47–50].

#### *3.3. MAP3K—MLK2/3, ASK1, TAK1*

MEKK1-4, DLK, TPL-2, TAO1/2, ASK1, MLK2/3, and TAK1 are common MAP3Ks involved in the activation pathway of JNK and p38, but the unique structure and subcellular distribution of these MAP3Ks reveal important roles in various cells and disease models. ASK1, MLK2/3, and TAK1 are widely studied and discussed in this review. ASK1 triggers cellular responses to redox stress and inflammatory cytokines [51,52] and plays important roles in innate immunity and viral infection [53]. ASK1 has the central kinase domain flanked on either side by coiled-coil domains. The N terminus of the kinase domain contains several regions with regulatory roles that bind to thioredoxin and TNF receptor-associated factors (TRAFs), which regulate the response of ASK1 to ROS and cytokines, respectively [54]. The N-terminal region of ASK1 has also been implicated in binding CIB1 to detect Ca2+-based stress signaling and in binding FBXO21 to trigger innate antiviral signaling [55,56]. The region C terminal to the kinase domain contains a 14-3-3 protein-binding site [57], followed by a region for constitutive oligomerization of ASK1 [58]. Under redox stress, thioredoxin dissociates and TRAF proteins associate with ASK1, which then tightly oligomerizes through its N-terminal coiled-coil (NCC) domain, promoting ASK1 activation and kinase activity via autophosphorylation of Thr845 in its kinase domain [59]. However, oxidative stress induces ubiquitination and subsequent degradation of activated ASK1. On the other hand, the deubiquitinating enzyme USP9X, which has ubiquitin-specific protease activity, interacts and antagonizes ubiquitination and subsequent degradation of activated ASK1 in H2O2-treated cells, resulting in the stabilization of activated ASK1 [60]. However, TNFAIP3 (A20), which has both ubiquitin ligase and deubiquitinase activities, inactivates ASK1 in fatty-acid-induced cells and ameliorates NASH [61]. Overexpression of A20 interacts with ASK1 and reduces stability and promotes the degradation of ASK1 through the ubiquitination process. Thus, overexpression of A20 lowers ASK1 level and preconditions cells to resist stress-induced JNK activation and cell death [62]. A20 is an acute response gene and overall effects of A20 will also depend on context and acute versus chronic disease. Contribution of A20 on JNK-SAB-ROS activation loop appears to be of minor importance. In addition to phosphorylation- and ubiquitination-mediated regulation of ASK1 in basal and response to oxidative stress, cFLIP competes for binding to TRAF binding domain of ASK1 and prevents ASK1 dimerization and activation [63]. However, ITCH promotes Lys48 ubiquitination and degradation of cFLIP. ITCH is activated by JNK [64]. JNK-ITCH-Ask1 signal activation axis does not affect TNF/GalN-induced apoptosis in contexts where SAB is deleted because depletion of SAB or MKK4/7 completely prevents TNFα-induced JNK activation and cell death, indicating requirement of MAP2K, JNK, and SAB to sustain ASK1 activation [7,12,65]. As mitochondria have thioredoxin-2, mitochondria localization of ASK1 and association with thioredoxin-2 has been proposed [66]. However, JNK activation has not been observed in mitochondria and further exploration is required. Another ASK1 regulator that has been recently identified is Caspase Recruitment Domain Protein 6 (CARD6) [67,68]. CARD6 associates with ASK1 and suppresses ASK1 phosphorylation activation and downstream JNK/p38 activation. In high-fat diet-induced fatty liver model, ASK1, MLK3, and TAK1 activation occurs. ASK1 phosphorylation is further increased by CARD6 deficiency but suppressed by CARD6 overexpression. TAK1 phosphorylation is not affected by CARD6, indicating selectivity of regulation. TAK1 is phosphorylated and activated via TLR/IL1 receptor and TRAFs [69]. Phosphorylated TAK1 activates IKK and MAP2K, leading to activation of NF-κB and JNK, respectively. Hepatocyte-specific deletion of TAK1 causes spontaneous hepatocyte death, suggesting hepatoprotective role of TAK1 [70], although further exploration is required.

MLK2/3 are redundant MAP3Ks regulated by small GTPases CDC42 and RAC1 [71]. Recently, JIP1 has been identified as a platform for interaction and signal integration of SRC tyrosine kinase, RAC GTPase, and MLKs for activation of MKK7 and JNK in free fatty acid-induced activation

model [19]. As JIP is a JNK-specific scaffold protein, the pathway selectively activates JNK but not p38 in free fatty acid-induced stress in MEF cells and diet-induced mouse models. The role of ASK1 was not examined in this free fatty acid-induced stress in MEF cell model. Indeed, JIP3/4 interacts with ASK1 but cannot mediate JNK activation [72]. The importance of JIP/MAP3K/MAP2K/MAPK signaling pathway in death receptor (TNF)-mediated MAP kinase activation requires further exploration.