A novel co-culture assay to assess anti-tumor CD8(+) T cell cytotoxicity via luminescence and multicolor flow cytometry

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8(+) T cells evidenced by in vitro CD8(+) T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFN gamma, and TNF alpha by CD8(+) T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8(+) T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations

Charged Particle and Conventional Radiotherapy:Current Implications as Partner for Immunotherapy

Simple Summary Immunotherapy provides the unprecedented opportunity to prolong the survival of cancer patients and even cure patients with previously untreatable malignancies. Preclinical and clinical studies show that standard photon-based radiotherapy and immunotherapy can synergize in order to promote both local and systemic anti-tumor immunity and that there is still ample room for improvement. Charged particle radiation is thought to have greater immunogenic potential compared to photon radiotherapy due to more lethal unrepaired damage, higher ionization density and thus more complex clustered DNA lesions. In this review, several factors determining the success of radiotherapy combined with immunotherapies, such as composition of the tumor, radiotherapy scheme and schedule, radiation dose, the type of radiation, are addressed. Furthermore, the theoretical basis, first pieces of evidences and new insights supporting a favorable immunogenicity profile of charged particle radiation are examined, including a depiction of best of knowledge for the immune-related responses triggered by charged particles and prospective clinical trials. Radiotherapy (RT) has been shown to interfere with inflammatory signals and to enhance tumor immunogenicity via, e.g., immunogenic cell death, thereby potentially augmenting the therapeutic efficacy of immunotherapy. Conventional RT consists predominantly of high energy photon beams. Hypofractionated RT regimens administered, e.g., by stereotactic body radiation therapy (SBRT), are increasingly investigated in combination with cancer immunotherapy within clinical trials. Despite intensive preclinical studies, the optimal dose per fraction and dose schemes for elaboration of RT induced immunogenic potential remain inconclusive. Compared to the scenario of combined immune checkpoint inhibition (ICI) and RT, multimodal therapies utilizing other immunotherapy principles such as adoptive transfer of immune cells, vaccination strategies, targeted immune-cytokines and agonists are underrepresented in both preclinical and clinical settings. Despite the clinical success of ICI and RT combination, e.g., prolonging overall survival in locally advanced lung cancer, curative outcomes are still not achieved for most cancer entities studied. Charged particle RT (PRT) has gained interest as it may enhance tumor immunogenicity compared to conventional RT due to its unique biological and physical properties. However, whether PRT in combination with immune therapy will elicit superior antitumor effects both locally and systemically needs to be further investigated. In this review, the immunological effects of RT in the tumor microenvironment are summarized to understand their implications for immunotherapy combinations. Attention will be given to the various immunotherapeutic interventions that have been co-administered with RT so far. Furthermore, the theoretical basis and first evidences supporting a favorable immunogenicity profile of PRT will be examined

Use of a Luciferase-Expressing Orthotopic Rat Brain Tumor Model to Optimize a Targeted Irradiation Strategy for Efficacy Testing with Temozolomide

Glioblastoma multiforme (GBM) is a common and aggressive malignant brain cancer with a mean survival time of approximately 15 months after initial diagnosis. Currently, the standard-of-care (SOC) treatment for this disease consists of radiotherapy (RT) with concomitant and adjuvant temozolomide (TMZ). We sought to develop an orthotopic preclinical model of GBM and to optimize a protocol for non-invasive monitoring of tumor growth, allowing for determination of the efficacy of SOC therapy using a targeted RT strategy combined with TMZ. A strong correlation (r = 0.80) was observed between contrast-enhanced (CE)-CT-based volume quantification and bioluminescent (BLI)-integrated image intensity when monitoring tumor growth, allowing for BLI imaging as a substitute for CE-CT. An optimized parallel-opposed single-angle RT beam plan delivered on average 96% of the expected RT dose (20, 30 or 60 Gy) to the tumor. Normal tissue on the ipsilateral and contralateral sides of the brain were spared 84% and 99% of the expected dose, respectively. An increase in median survival time was demonstrated for all SOC regimens compared to untreated controls (average 5.2 days, p < 0.05), but treatment was not curative, suggesting the need for novel treatment options to increase therapeutic efficacy

Lymphocyte-Sparing Radiotherapy: The Rationale for Protecting Lymphocyte-rich Organs When Combining Radiotherapy With Immunotherapy

There is now strong clinical and preclinical evidence that lymphocytes, e.g. CD8(+) T cells, are key effectors of immunotherapy and that irradiation of large blood vessels, the heart, and lymphoid organs (including nodes, spleen, bones containing bone marrow, and thymus in children) causes transient or persistent lymphopenia. Furthermore, there is extensive clinical evidence, across multiple cancer sites and treatment modalities, that lymphopenia correlates strongly with decreased overall survival. At the moment, we lack quantitative evidence to establish the relationship between dose-volume and dose-rate to critical normal structures and lymphopenia. Therefore, we propose that data should be systematically recorded to characterize a possible quantitative relationship. This might enable us to improve the efficacy of radiotherapy and develop strategies to predict and prevent treatment-related lymphopenia. In anticipation of more quantitative data, we recommend the application of the principle of As Low As Reasonably Achievable (ALARA) to lymphocyte rich regions for radiotherapy treatment planning to reduce the radiation doses to these structures, thus moving toward “Lymphocyte-Sparing Radiotherapy”

Hypoxia PET Imaging with [18F]-HX4-A Promising Next-Generation Tracer

Hypoxia—a common feature of the majority of solid tumors—is a negative prognostic factor, as it is associated with invasion, metastasis and therapy resistance. To date, a variety of methods are available for the assessment of tumor hypoxia, including the use of positron emission tomography (PET). A plethora of hypoxia PET tracers, each with its own strengths and limitations, has been developed and successfully validated, thereby providing useful prognostic or predictive information. The current review focusses on [18F]-HX4, a promising next-generation hypoxia PET tracer. After a brief history of its development, we discuss and compare its characteristics with other hypoxia PET tracers and provide an update on its progression into the clinic. Lastly, we address the potential applications of assessing tumor hypoxia using [18F]-HX4, with a focus on improving patient-tailored therapies