Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development.

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α₂ associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response—cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.

Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α₁ after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α₁ after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening.

Background: Photodynamic therapy (PDT) leading to immunogenic cell death (ICD) may serve as a promising basis for the development of antitumor therapeutic strategies. However, the mechanisms of action of photoinduced ICD in primary tumor cultures, including human glioma, remain unexplored. Methods: In the present study, the features of regulated cell death induced by photodynamic therapy using a previously described ICD inducer, porphyrazine III (pz III), were investigated. Cell death was studied in 7 primary cultures of high-grade human gliomas (astrocytomas, oligodendrogliomas, and glioblastomas). Results: Accumulation of porphyrazine III was observed in the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and mitochondria; however, the distribution of the photosensitizer varied across different cultures. A narrow concentration window of porphyrazine III was established to effectively reach IC85, primarily inducing ferroptosis with contributions from apoptosis and necroptosis accompanied by superoxide anion generation and mitochondrial dysfunction. Conclusions: Given the immunogenic potential of ferroptosis, apoptosis and necroptosis we hypothesize that the induction of PDT using porphyrazine III in glioma will trigger immunogenic cell death.

Background/Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor with dismal prognosis and limited treatment options. Immunotherapy, including personalized approaches using tumor-infiltrating lymphocytes (TILs) and allogeneic natural (NK) or engineered killer cells (chimeric antigen receptor NK, NK-CAR), and oncolytic viruses (OV), has shown some potential in GBM. Combining different therapeutic strategies may enhance treatment efficacy. Here, we present a xenograft GBM mouse model with multiparametric detection for various immunotherapy research applications. Methods: In a xenograft GBM NOD-Prkdcs scid Il2rgem1/Smoc (NSG) mouse model based on orthotopic transplantation of patient-derived GBM cultures retaining tumor heterogeneity, intravenous and intratumor immunotherapeutic interventions by TIL and OV therapy were performed. Xenograft engraftment was evaluated using intravital MRI; delivery of OV and TILs to the tumor and changes in the tumor and peritumoral space were assessed using intravital confocal microscopy; and metabolic and structural changes in the tumor and peritumoral environment were assessed via fluorescence lifetime imaging microscopy (FLIM) and optical coherence tomography (OCT). The intravital imaging data were compared with the results of preliminary and final histological and immunocytochemical data. Results: Both OV and TILs demonstrated tumor-specific targeting and delivery across the blood–brain barrier. Further, we showed that in this model the xenograft response to both therapeutic treatments can be assessed using FLIM and OCT. Conclusions: Overall, this work presents an optimized mouse model suitable for assessing the effect of combined TIL immunotherapy and OV on GBM in translational studies.