Citations

Abstract Introduction Immunotherapy approaches against glioblastoma (GBM) such as immune checkpoint inhibitors (ICI), dendritic cell vaccination, and CAR-T have failed to durably cure most patients. Studies analyzing tumors from ICI responders show that abundance of CD4 and CD8 T cells prior to initiation of therapy predicts responsiveness. Thus, targeting the microenvironment to promote CD4 activity may synergize with ICI. The glioblastoma microenvironment contains high proportions of monocyte-derived macrophages which have potent immunosuppressive capabilities. Characterizing and targeting these immunosuppressive programs is a promising strategy for designing effective combination immunotherapies for glioblastoma patients. Methods We analyzed human glioblastoma snRNAseq datasets to find signaling receptors associated with myeloid immunosuppression and employed murine models of glioblastoma for functional validation. Results By analyzing patient-derived -omics datasets, we identified an understudied TNF-superfamily receptor, Fn14, as being upregulated in glioblastoma-associated myeloid cells with immunosuppressive gene signatures. Using in vitro approaches, we found that Fn14 activation in the tumor microenvironment can drive cytokines implicated in GBM immunosuppression such as IL6, IL1B, and IL8. Thus, Fn14 may serve as a surface marker for microenvironment-remodeling myeloid cells recruited to the tumor from peripheral circulation. Furthermore, in murine models of glioblastoma, we discovered Fn14 KO among glioblastoma-associated myeloid cells increases conventional dendritic cell type 1 (cDC1) and Th1 CD4+ T cell abundance in the tumor microenvironment and significantly prolongs survival. Conclusion These results suggest that blocking Fn14 in glioblastoma-associated myeloid cells can enhance Th1 immunity. Thus, Fn14 may represent a promising dual therapy target. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Background Fibroblast growth factor-inducible 14 (Fn14) belongs to the TNFR superfamily. Fn14 overexpression can drive receptor-autonomous signaling, increase both cell invasion and tumor-associated macrophages/microglia (TAMMs) recruitment, and correlates with reduced survival in glioblastoma (GBM) patients and rat gliomas. While prior studies report Fn14 expression in non-tumor cells within the GBM tumor microenvironment (TME), their relative contributions to glioma pathobiology remain unclear. Methods Using tumor-host pairings of Fn14-positive and -knockout (-KO) cells and mice, we examined the role of Fn14 in glioma biology. Mouse glioma and human GBM datasets were analyzed at the cellular, protein, and transcriptomic levels to assess Fn14-associated changes in the glioma TME and survival outcomes. Results Fn14 was found to be highly expressed in tumor cells and TAMMs in human GBM and 2 well-characterized murine glioma models. Fn14 KO in both tumor and host cells increased overall survival. Notably, this survival benefit was greater in the glioma model characterized by a more immunologically activated TME. Immunophenotyping revealed that Fn14 loss reshapes the tumor-immune landscape, reducing the presence of immunosuppressive macrophages and exhausted T-cells, suggesting that Fn14 modulates both innate and adaptive immune responses. These findings were supported by analyses of human GBM datasets, where high Fn14 expression correlated with immunosuppressive shifts and poor patient responses to immune checkpoint inhibitor therapy. Conclusions This study provides the first description of the contributions of both tumor- and host-derived Fn14 expression to tumor immunity and survival and identifies Fn14 as an important mediator of innate and adaptive immune responses in gliomas.

TPS2094 Background: Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating tumor (ct) and cell-free (cf) DNA levels in blood due to the blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with the development and use of biomarker-driven systemic therapies. Low intensity focused ultrasound combined with intravenously administered microbubble oscillators (MB-FUS), leads to non-invasive BBB opening. This trial aims to evaluate the utility of LIFU for bolstering blood ctDNA and cfDNA for enhance liquid biopsy in patients with GBM. Methods: LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial evaluating safety and technical efficacy of transcranial MR-guided MB-FUS for increasing blood ctDNA and cfDNA levels in adults, aged 18-80 years with GBM. Patients with suspected GBM planned for tumor biopsy or resection at 17 centers in US and Canada are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication, after which MR-guided BBB opening using a 220 kHz device, with 1024-element phased array transducer, is performed with real-time acoustic feedback control for effective cavitation. Pre- and post-procedure, phlebotomy and MRI brain are done. Patients are offered optional 2 nd procedure during adjuvant chemotherapy phase if willing. Primary efficacy endpoint is correlation between biomarker patterns in tumor tissue collected during surgery/biopsy and blood collected following MB-FUS procedure. Confirmatory secondary efficacy endpoint is ratio between greatest yield of cfDNA in blood post-MB-FUS compared to cfDNA level in blood pre-MB-FUS. The primary study hypothesis is that agreement rate on biomarker pattern between resected/biopsied tumor tissue and blood is > 70%. The secondary hypothesis is that MB-FUS BBBO leads to a ≥2-fold rise in blood cfDNA. Assuming the true agreement rate expected is 89%, a sample of N = 50 patients will provide 90% power to meet the primary endpoint (Exact test, Binomial Proportion, one-sided Alpha = 0.025). Exploratory endpoints include (1) sensitivity of detection of known specific somatic mutations in ctDNA from blood samples collected before and after MB-FUS, (2) estimation of ctDNA levels in samples collected at 30-minutes, 1-hour, 2-hour, and 3-hour post-MB-FUS to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBB opening and ctDNA-based biomarkers from post-MB-FUS blood samples, (4) biomarker correlation between plasma cfDNA sampled during adjuvant chemotherapy phase and tumor tissue harvested at surgery. Patient enrollment commenced in 2022 and is ongoing (NCT05383872). Clinical trial information: NCT05383872 .

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by insufficient requisite circulating-tumor (ct) and cell-free (cf) DNA in blood due to blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with biomarker-driven systemic therapies. Real-time image-guided low-intensity focused ultrasound (LIFU) plus IV microbubble oscillators, leads to non-invasive BBB disruption. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial investigating safety and technical efficacy of LIFU-based BBB disruption for increasing blood ctDNA and cfDNA levels in adult GBM patients (aged 18-80 years). Patients with suspected GBM planned for biopsy/resection at ten US centers are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication effects, after which MR-guided BBB disruption using 220kHz LIFU device is performed with real-time acoustic-feedback-control for effective microbubble activation. Pre- and post-LIFU, phlebotomy and MRI brain are performed. Primary study endpoint is defined, per subject, as ratio between their blood cfDNA level 1-hour post-LIFU procedure compared to blood cfDNA pre-procedure. Primary study hypothesis is that BBB disruption leads to ≥2-fold rise in blood cfDNA. Secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in blood cfDNA sample obtained 1-hour post-LIFU and biomarker pattern in tumor tissue obtained later. Trial is powered to evaluate both primary and secondary hypotheses. Based on assumed true agreement rate of 91% and one-sided alpha=0.025, an exact test for binomial proportions provides N=50 with 84% power for secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of specific somatic mutations in ctDNA from blood collected pre- and post-LIFU, (2) ctDNA estimation in samples collected at 30-minutes, 1-hour, 2-hour, and 3-hour post-LIFU to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBB disruption and ctDNA-based biomarkers from post-LIFU blood samples. Patient enrollment commenced in 2022 and remains ongoing (NCT05383872).

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating tumor (ct) and cell-free (cf) DNA levels in blood due to the blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with the development and use of biomarker-driven systemic therapies. Real-time image-guided low intensity focused ultrasound (LIFU) combined with intravenously administered microbubble oscillators, leads to non-invasive disruption of the BBB. This trial aims to evaluate the utility of LIFU for boosting blood ctDNA and cfDNA for enhanced liquid biopsy in GBM patients. MATERIAL AND METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial evaluating safety and technical efficacy of LIFU-based BBB disruption for increasing blood ctDNA and cfDNA levels in adults, aged 18-80 years with GBM. Patients with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication effects, after which MR-guided BBB disruption using a 220 kHz LIFU device is performed with real-time acoustic feedback control for effective microbubble activation. Before and after procedure, phlebotomy and MRI brain are performed to evaluate outcomes. Primary study endpoint is defined, per subject, as ratio between their cfDNA level in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. Primary study hypothesis is that BBB disruption with LIFU leads to a ≥2-fold rise in blood cfDNA. The secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in blood cfDNA sample obtained 1-hour post-LIFU and the biomarker pattern in tumor tissue obtained later. The trial has been powered to evaluate both primary and secondary hypotheses. Based on an assumed true agreement rate of 91% and a one-sided alpha of 0.025, an exact test for binomial proportions provides a sample of N = 50 with 84% power for the secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of known specific somatic mutations in ctDNA from blood samples collected before and after LIFU, (2) estimation of ctDNA levels post-LIFU in samples collected at 30 minutes, 1 hour, 2 hour, and 3 hour to determine the time of greatest yield, (3) correlation of MRI parameters related to grading of BBB disruption and ctDNA-based biomarkers from post-LIFU blood samples. RESULTS Patient enrollment commenced in 2022, and 22 patients have been recruited by 05/2024. CONCLUSION Findings from this trial will be critical to liquid biopsy in GBM (Clinical trial information: NCT05383872).

TPS2098 Background: Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating tumor (ct) and cell-free (cf) DNA levels in blood due to the blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with the development and use of biomarker-driven systemic therapies. Real-time image-guided low intensity focused ultrasound (LIFU) combined with intravenously administered microbubble oscillators, leads to non-invasive disruption of the BBB. This trial aims to evaluate the utility of LIFU for boosting blood ctDNA and cfDNA for enhanced liquid biopsy in GBM patients. Methods: LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial evaluating safety and technical efficacy of LIFU-based BBB disruption for increasing blood ctDNA and cfDNA levels in adults (aged 18-80 years) with GBM. Patients with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication effects, after which MR-guided BBB disruption using a 220 kHz LIFU device is performed with real-time acoustic feedback control for effective microbubble activation. Before and after procedure, phlebotomy and MRI brain are performed to evaluate outcomes. Primary study endpoint is defined, per subject, as ratio between their cfDNA level in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. Primary study hypothesis is that BBB disruption with LIFU leads to a ≥2-fold rise in blood cfDNA. The secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in blood cfDNA sample obtained 1-hour post-LIFU and the biomarker pattern in tumor tissue obtained later. The trial has been powered to evaluate both primary and secondary hypotheses. Based on an assumed true agreement rate of 91% and a one-sided alpha of 0.025, an exact test for binomial proportions provides a sample of N = 50 with 84% power for the secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of known specific somatic mutations in ctDNA from blood samples collected before and after LIFU, (2) estimation of ctDNA levels post-LIFU in samples collected at 30 minutes, 1 hour, 2 hour, and 3 hour to determine the time of greatest yield, (3) correlation of MRI parameters related to grading of BBB disruption and ctDNA-based biomarkers from post-LIFU blood samples. Patient enrollment commenced in 2022 and 22 patients have been recruited by January 2024. Clinical trial information: NCT05383872 .

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6867.

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) blood levels in due to the blood-brain barrier (BBB). Real-time image-guided low-intensity focused ultrasound (LIFU) with intravenously administered microbubble oscillators non-invasively causes BBB disruption (BBBD). This clinical trial aimed to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating the safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged 18-80 years with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being included. Patients are administered intravenous oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using 220 kHz LIFU device is performed, with real-time acoustic feedback for effective sonication. Pre- and post-procedure, phlebotomies, and MRI brain are performed. The primary study endpoint is defined, per subject, as the ratio between cfDNA levels in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% concordance between biomarker patterns in the cfDNA sample from 1-hour post-LIFU sample and tumor tissue obtained later. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels in samples collected at 30 minutes, 1 hour, 2 hours, and 3 hours post-LIFU to determine the time of greatest yield, (3) correlation of MRI parameters related to BBBD grading and biomarkers in cfDNA from post-LIFU samples. Patient enrollment commenced in 2022, and 13 patients have been recruited by 06/09/2023. Clinical trial information: NCT05383872.

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) levels in blood due to the blood-brain barrier (BBB). This results in challenges to the identification of blood-based biomarkers and the development of novel biomarker-driven systemic therapies. Real-time image-guided low intensity focused ultrasound (LIFU) combined with intravenously administered microbubble oscillators non-invasively causes BBB disruption (BBBD). This clinical trial aimed to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. METHODS LIBERATE is a prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating the safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged 18-80 years with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being included. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered intravenous oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using a 220 kHz LIFU device is performed with real-time acoustic feedback for effective cavitation. Before and after the procedure, phlebotomies and MRI brain are performed to evaluate outcomes. The primary study endpoint is defined, per subject, as the ratio between their cfDNA level in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to a ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in cfDNA sample from 1-hour post-LIFU sample and biomarker pattern in tumor tissue obtained later. The trial has been powered to evaluate both primary and secondary hypotheses. Based on an assumed true agreement rate of 91% and a one-sided alpha of 0.025, an exact test for binomial proportions provides a sample of N=50 with 84% power for the secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels post-LIFU in samples collected at 30 minutes, 1 hour, 2 hours, and 3 hours to determine the time of greatest yield, (3) correlation of MRI parameters related to grading of BBBD and biomarkers positive in cfDNA from post-LIFU blood samples. RESULTS Patient enrollment commenced in 2022, and 8 patients have been recruited by 04/11/2023. CONCLUSION Findings from this trial will be critical to liquid biopsy in GBM (Clinical trial information: NCT05383872).

TPS2084 Background: Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) levels in blood due to the blood-brain barrier (BBB). This results in challenges to the identification of blood-based biomarkers and the development of novel biomarker-driven systemic therapies. Real-time image-guided low intensity focused ultrasound (LIFU) combined with IV microbubble oscillators (DEFINITY), non-invasively causes BBB disruption (BBBD). This clinical trial aims to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. Methods: LIBERATE is a prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged >18-80 years with suspected GBM planned for tumor biopsy or resection at eleven centers in North America are being included. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using a 220 kHz LIFU device is performed with real-time acoustic feedback for effective cavitation. Before and after procedure, phlebotomies and MRI brain are performed to evaluate outcomes. The primary study endpoint is defined, per subject, as the ratio between their cfDNA level in blood 1-hour post-LIFU compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in cfDNA sample from 1-hour post-LIFU sample and biomarker pattern in tumor tissue obtained later. The trial has been powered to evaluate both primary and secondary hypotheses. Based on an assumed true agreement rate of 91% and a one-sided alpha of 0.025, an exact test for binomial proportions provides a sample of N=50 with 84% power for the secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels post-LIFU in samples collected at 30 minutes, 1 hour, 2 hour, and 3 hour to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBBD and biomarkers positive in cfDNA from post-LIFU blood samples. Patient enrollment commenced in 2022 and 7 patients have been recruited by 02/13/2023. Clinical trial information: NCT05383872 .