A Cellular Ground Truth to Develop MRI Signatures in Glioma Models by Correlative Light Sheet Microscopy and Atlas-Based Coregistration.

Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.SIGNIFICANCE STATEMENT We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.

Assessment of Tumor Cell Invasion and Radiotherapy Response in Experimental Glioma by Magnetic Resonance Elastography.

BACKGROUND: Gliomas are highly invasive brain neoplasms. MRI is the most important tool to diagnose and monitor glioma but has shortcomings. In particular, the assessment of tumor cell invasion is insufficient. This is a clinical dilemma, as recurrence can arise from MRI-occult glioma cell invasion. HYPOTHESIS: Tumor cell invasion, tumor growth and radiotherapy alter the brain parenchymal microstructure and thus are assessable by diffusion tensor imaging (DTI) and MR elastography (MRE). STUDY TYPE: Experimental, animal model. ANIMAL MODEL: Twenty-three male NMRI nude mice orthotopically implanted with S24 patient-derived glioma cells (experimental mice) and 9 NMRI nude mice stereotactically injected with 1 µL PBS (sham-injected mice). FIELD STRENGTH/SEQUENCE: 2D and 3D T2-weighted rapid acquisition with refocused echoes (RARE), 2D echo planar imaging (EPI) DTI, 2D multi-slice multi-echo (MSME) T2 relaxometry, 3D MSME MRE at 900 Hz acquired at 9.4 T (675 mT/m gradient strength). ASSESSMENT: Longitudinal 4-weekly imaging was performed for up to 4 months. Tumor volume was assessed in experimental mice (n = 10 treatment-control, n = 13 radiotherapy). The radiotherapy subgroup and 5 sham-injected mice underwent irradiation (3 × 6 Gy) 9 weeks post-implantation/sham injection. MRI-/MRE-parameters were assessed in the corpus callosum and tumor core/injection tract. Imaging data were correlated to light sheet microscopy (LSM) and histology. STATISTICAL TESTS: Paired and unpaired t-tests, a P-value ≤0.05 was considered significant. RESULTS: From week 4 to 8, a significant callosal stiffening (4.44 ± 0.22 vs. 5.31 ± 0.29 kPa) was detected correlating with LSM-proven tumor cell invasion. This was occult to all other imaging metrics. Histologically proven tissue destruction in the tumor core led to an increased T2 relaxation time (41.65 ± 0.34 vs. 44.83 ± 0.66 msec) and ADC (610.2 ± 12.27 vs. 711.2 ± 13.42 × 10-6 mm2/s) and a softening (5.51 ± 0.30 vs. 4.24 ± 0.29 kPa) from week 8 to 12. Radiotherapy slowed tumor progression. DATA CONCLUSION: MRE is promising for the assessment of key glioma characteristics. EVIDENCE LEVEL: NA TECHNICAL EFFICACY: Stage 2.

Tumor biomechanics as a novel imaging biomarker to assess response to immunotherapy in a murine glioma model.

Glioblastoma is the most common and aggressive primary malignant brain tumor with poor prognosis. Novel immunotherapeutic approaches are currently under investigation. Even though magnetic resonance imaging (MRI) is the most important imaging tool for treatment monitoring, response assessment is often hampered by therapy-related tissue changes. As tumor and therapy-associated tissue reactions differ structurally, we hypothesize that biomechanics could be a pertinent imaging proxy for differentiation. Longitudinal MRI and magnetic resonance elastography (MRE) were performed to monitor response to immunotherapy with a toll-like receptor 7/8 agonist in orthotopic syngeneic experimental glioma. Imaging results were correlated to histology and light sheet microscopy data. Here, we identify MRE as a promising non-invasive imaging method for immunotherapy-monitoring by quantifying changes in response-related tumor mechanics. Specifically, we show that a relative softening of treated compared to untreated tumors is linked to the inflammatory processes following therapy-induced re-education of tumor-associated myeloid cells. Mechanistically, combined effects of myeloid influx and inflammation including extracellular matrix degradation following immunotherapy form the basis of treated tumors being softer than untreated glioma. This is a very early indicator of therapy response outperforming established imaging metrics such as tumor volume. The overall anti-tumor inflammatory processes likely have similar effects on human brain tissue biomechanics, making MRE a promising tool for gauging response to immunotherapy in glioma patients early, thereby strongly impacting patient pathway.

In vivo nanoparticle-based T cell imaging can predict therapy response towards adoptive T cell therapy in experimental glioma.

Rationale: Intrinsic brain tumors, such as gliomas are largely resistant to immunotherapies including immune checkpoint blockade. Adoptive cell therapies (ACT) including chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cell therapy targeting glioma-associated antigens are an emerging field in glioma immunotherapy. However, imaging techniques for non-invasive monitoring of adoptively transferred T cells homing to the glioma microenvironment are currently lacking. Methods: Ultrasmall iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Here, we develop a protocol for efficient ex vivo labeling of murine and human TCR-transgenic and CAR T cells with iron oxide NPs. We assess labeling efficiency and T cell functionality by flow cytometry and transmission electron microscopy (TEM). NP labeled T cells are visualized by MRI at 9.4 T in vivo after adoptive T cell transfer and correlated with 3D models of cleared brains obtained by light sheet microscopy (LSM). Results: NP are incorporated into T cells in subcellular cytoplasmic vesicles with high labeling efficiency without interfering with T cell viability, proliferation and effector function as assessed by cytokine secretion and antigen-specific killing assays in vitro. We further demonstrate that adoptively transferred T cells can be longitudinally monitored intratumorally by high field MRI at 9.4 Tesla in a murine glioma model with high sensitivity. We find that T cell influx and homogenous spatial distribution of T cells within the TME as assessed by T2* imaging predicts tumor response to ACT whereas incomplete T cell coverage results in treatment resistance. Conclusion: This study showcases a rational for monitoring adoptive T cell therapies non-invasively by iron oxide NP in gliomas to track intratumoral T cell influx and ultimately predict treatment outcome.

T cell-independent eradication of experimental glioma by intravenous TLR7/8-agonist-loaded nanoparticles.

Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically "cold" tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a ß-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.