Molecular imaging biomarkers for cell-based immunotherapies

Recent advances have demonstrated that blockade of immune checkpoints is one of the most promising approaches for activating therapeutic antitumor immunity [8]. Immune checkpoints are the receptor-ligand pairs on the cell surface that are involved in regulating T-cell activation.

It is now established that tumors utilize certain immune-checkpoint pathways as a mechanism of immune resistance against T cells that are specific for tumor antigens. Since many of the immune checkpoints involve ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. Immunotherapeutics based on antibodies of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) [17, 18] and programmed cell death protein-1 (PDCD1/PD1) are showing promising results of antitumor immunity [19, 20]. In fact, the immunomodulatory monoclonal antibody of CTLA4, Ipilimumab, is the first Food and Drug Administration (FDA) approved immunotherapeutic agent for treating cancer [10, 21]. More recently, Nivolumab and Pembrolizumab, humanized IgG4 antibodies, which block PD-1 and inhibits its interaction with PD-L1 and PD-L2 have also been approved as immunotherapeutic agents for treatment of cancer by the US FDA [22‐24].

Adoptive T-cell therapies include expanded autologous T cells and T cells with engineered T-cell receptors (TCRs) and chimeric antigen receptors (CARs) [25, 26]. Specifically, tumor-infiltrating lymphocytes (TILs) are isolated from tumor biopsies and expanded before being reinfused into the patient, based on the premise that these TILs are tumor cell specific. The most effective T-cell therapies explored in clinical trials currently focus on leukemia, but are also used to treat patients with solid tumors.

Cytokines play important roles in the body’s normal immune responses and also in the immune system’s ability to respond to cancer. Interferons and interleukins are two main types of cytokines used to treat cancer [13, 14]. These cytokines bind to their receptors on T cells, and stimulate the activation and proliferation of T cells and downstream production of more cytokines [13, 14].

These vaccines stimulate an active immune response against tumor by eliciting adaptive immunity through the patient’s own immune system. After injection of peptide or protein vaccines, the body’s antigen presenting cells (APCs) process vaccines as antigenic fragments to be recognized and stimulate the patient’s naïve T cells, which in turn may stimulate an endogenous immune response against cancer [15, 16].

While these immunotherapy methods provide tremendous hope for patients, they also present significant challenges. Treatment with immunotherapies is showing new patterns of treatment response and side effects. Specifically, after immunotherapy the response can be manifested different ways: (1) a decrease in size of known tumors without the presence of new tumor after completion of treatment, (2) clinically stable disease after completion of treatment and significantly delayed decrease in tumor size, (3) new or enlarging tumors observed soon after completion of treatment, which may not reflect disease progression, preceding a later decrease in tumor burden (4) autoimmune-mediated toxic effects that could be mistaken for metastatic disease or misdiagnosed as a non-treatment-related process and delay appropriate clinical management [27].

Currently, there are no robust biomarkers to identify the patients who will most likely benefit from these treatments. In the absence of a predictive biomarker, many patients may receive these expensive treatments without any benefit. These unconventional treatment response patterns and the wide range of autoimmune toxic effects make it rather challenging to monitor the effects of immunotherapies using Response Evaluation Criteria in Solid Tumors (RECIST) [28] criteria, which are based on the conventional anatomical imaging by computed tomography and magnetic resonance imaging (MRI) [29]. Hence, there is need for robust technology, which not only characterizes the immune microenvironment of tumors but also screen for patients who can potentially respond to immunotherapies. Imaging methods targeting T cell metabolism have the potential for providing molecular imaging biomarkers to assess immunotherapy response. To develop molecular imaging biomarkers, understanding the T cell metabolism and its changes upon activation are crucial.

Currently there are two major methods employed in measuring lactate in vivo. One is traditional magnetic resonance spectroscopy (MRS; both ¹H and ¹³C) [44‐46], which has been used to measure both static lactate levels and dynamic changes. However, these are limited by inadequate sensitivity and spatial resolution. The other method involves infusion of dynamic nuclear polarized (DNP)¹³C-labeled pyruvate, which provides greater than 10,000-fold signal enhancement compared to conventional MRS [47‐49]. Despite its high sensitivity, this method only probes fast kinetics (<1 min) of lactate turnover from ¹³C-labeled pyruvate and it requires special equipment and complex modeling for data analysis.

Recently, MRI method based on lactate CEST (LATEST) [42] to image lactate was described. LATEST method utilizes standard proton MRI and requires neither ¹³C labeled pyruvate nor DNP polarization. The feasibility of measuring LATEST in vivo was demonstrated in a lymphoma tumor model (Fig. 3), and in human skeletal muscle [42]. Dynamic changes in LATEST are reported in tumors pre- and post-infusion of pyruvate, and in exercising human skeletal muscle [42]. LATEST measurements are compared to lactate measured with multiple quantum filtered proton MRS [42]. LATEST provides over two orders of higher sensitivity compared to the ¹H MRS based lactate detection methods.

It was reported that lactic acid produced by the tumor cells blunts the tumor immunosurveillance by T and natural killer cells. This implies that the higher levels of lactate in tumor cells may adversely affect the immunotherapy and basal levels of lactate itself might give a clue regarding the response to immunotherapy [50]. In the context of immunotherapy, pre-therapy LATEST images provide the basal levels of lactate in tumor regions which are largely glycolytic and produce more lactate. Immediately, post-therapy (12–24 h), if the immune cells have identified receptors on the tumors cell surface and get activated then the T_EFF cells switch their metabolism to glycolysis and begin to proliferate rapidly and dump lots of lactate into the tumor microenvironment (at this time tumor cells may be still be producing lactate, although with a different/slower rate). This rapid increase in lactate can be measured using LATEST. In addition to this rapid increase in LATEST, T cells activation may also lead to side effects associated with autoimmunity. As the T cell rapidly proliferates, it may lead to an increase in the size of the T cells mass in the tumor region, which is often mistaken for tumor growth. This elevation in the lactate levels remains until the T cells completely destroy the tumor cells and then levels begin to drop, as the T_EFF cells die and convert to T_M cells, to basal values.

On the other hand, if the immune cells do not get activated then their metabolism remains OXPHOS and there would not be any change in the lactate levels due to immune cells and as the tumor cells are continuously proliferating, lactate levels and tumor size increase gradually. So the kinetics of the lactate measured shed light on the therapeutic efficacy.

The slopes of the lactate concentration vs. time curves, especially hours after the treatment, will serve as a measure of the response. Response to therapy is expected to produce a steeper slope in the curve than no response.

Higher concentration of glutamate, alanine and creatine during the T cell proliferation in response to immunotherapy can also be monitored using CEST. Studies have shown that the changes in these metabolites level in cancer tissue can be monitored non-invasively through CEST. Different CEST based approaches (GluCEST, glutamate; AlaCEST, alanine; CrCEST, creatine) have been developed to image these metabolites in vivo. In addition, another CEST method, amide proton transfer (APT), which primarily depends on the mobile protein content, has been shown to be useful in discriminating between tumor regrowth and radiation necrosis [51]. It has been shown that the glutamate released by the dendritic cells mediates the T cell activation/proliferation [52]. Higher expression of glutamate metabotropic receptor on activated T cells further confirms the role of the glutamate in T cells mediated immunity [52]. The increase in alanine concentration in an in vitro stimulated T cell line (Fig. 2) suggests that the activation of T cells result in more alanine synthesis. Changes in the in vivo glutamate, alanine and creatine level post-immunotherapy as measured by CEST may also serve as potential biomarkers to evaluate the treatment response.