isct: Preclinical Imaging of the Immune System

Multiscale Microscopy Of Cellular Immunotherapies

Immunotherapy is an emerging first-line therapy for advanced cancer with the potential to achieve long-lasting regression and cure. Most solid tumors respond to immunotherapy to varying degrees, but the majority of patients experience resistance in tumor subregions followed by relapse. The main effector cells that mediate tumor control are cytotoxic T lymphocytes (CTLs), which kill cancer cells in a cell contact- and antigen-specific manner. However, although CTLs are observed to infiltrate tumors in patients, their ability to control tumor growth is often insufficient. Consequently, immunotherapeutic strategies aim to activate and expand tumor antigen-specific CTLs and further improve their ability to kill within the tumor. Current therapeutic approaches include inhibition of immune checkpoints to silence inhibitory receptors, vaccination with dendritic cells to stimulate endogenous antitumor immune activation and adoptive transfer of patient-derived, genetically modified T cells to enhance the number of tumor-specific cells with strong killing capacity. While each approach has demonstrated experimental and clinical success, no individual or combined strategy has achieved sufficient efficacy in a majority of patients.

Microenvironment-Controlled Immune Function

The clinical success of adoptive T cell transfer in solid tumors appears to be limited by several immunosuppressive barriers imposed by the tumor microenvironment. Transferred CTLs are excluded from tumor lesions or become dysfunctional upon entry into the tumor, interaction with tumor cells is too short-lived to reach full cytotoxic potential, and suppressive immune cells directly or indirectly interfere with CTL activity. Furthermore, in the same patient, distinct microenvironments, such as those at the primary tumor site and metastatic lesions, differ in their response to therapy. In particular, bone metastases are typically resistant to immunotherapy despite successful immune infiltration of the primary lesion. The factors in the bone marrow that inhibit the efficiency of cell-based immunotherapies and the biomarkers that can be used to detect therapeutic failure in a patient at an early stage are only incompletely understood. In ongoing studies, we aim to correlate information from macroscopic imaging (PET/MRI) with cellular and molecular profiling to identify immunosuppressive signatures in bone that can serve as biomarkers for noninvasive therapy monitoring as well as target structures for new therapeutic approaches.

Synergy Of Macro- and Micorscale Imaging

Experimental approaches that investigate the immunotherapy response in small animals are mostly based on static images (e.g., immunohistochemistry) or macroscopic imaging (e.g., bioluminescence, CT, PET, MRI). Although informative, these strategies lack sensitivity or temporal and spatial resolution to characterize dynamic and reciprocal interactions between tumor cells, the microenvironment and immune effector cells, which occur in defined tissue niches. Mechanistic, 3D and time-resolved insights into the positioning and function of single cells during therapy, their adjacent environment, and tumor-stroma interactions have been made possible by technological advances in intravital multiphoton microscopy (iMPM). Intravital microscopy is suitable for accessing niches that mediate tumor progression, including cell growth, motility and invasion, remodeling of the stroma, neovessel anatomy and function, and regulation of molecular signaling events. Similarly, iMPM is capable of capturing all functionally relevant steps of adoptive T cell therapy, including the arrival of transferred cells in the lesion, early effector function and induction of tolerance and CTL exhaustion (Figure 1). Thus, microscopic imaging provides mechanistic insight at the single cell level and is essential for developing the bases for novel treatment approaches. Macroscopic imaging allows the monitoring of systemic immune effects and the early identification of therapy efficacy or potential side effects in patients. Ongoing research addresses how both imaging scales can be combined to deliver synergistic information about immune function during therapy.

Figure 1: Intravital multiphoton microscopy allows the monitoring of immune cell function and tumor therapy response at the single-cell level (A). Visualization of essential steps during immunotherapy, such as immune cell arrival through blood vessels and infiltration and positioning of immune cells within the tumor, as well as single cell dynamics, such as immune cell proliferation and tumor immune cell contact followed by tumor cell apoptosis (B). Therefore, rate-limiting steps and tumor resistance niches can be identified, which provide rationales for novel treatment strategies and synergistic therapy combinations.