Tumor Treating Fields (TTFields), an approved therapy for glioblastoma (GBM) and malignant mesothelioma, employ noninvasive application of low-intensity, intermediate-frequency, alternating electric fields to disrupt the mitotic spindle, leading to chromosome missegregation and apoptosis. Emerging evidence suggests that TTFields may also induce inflammation. However, the mechanism underlying this property and whether it can be harnessed therapeutically are unclear. Here, we report that TTFields induced focal disruption of the nuclear envelope, leading to cytosolic release of large micronuclei clusters that intensely recruited and activated 2 major DNA sensors — cyclic GMP-AMP synthase (cGAS) and absent in melanoma 2 (AIM2) — and their cognate cGAS/stimulator of interferon genes (STING) and AIM2/caspase 1 inflammasomes to produce proinflammatory cytokines, type 1 interferons (T1IFNs), and T1IFN-responsive genes. In syngeneic murine GBM models, TTFields-treated GBM cells induced antitumor memory immunity and a cure rate of 42% to 66% in a STING- and AIM2-dependent manner. Using single-cell and bulk RNA sequencing of peripheral blood mononuclear cells, we detected robust post-TTFields activation of adaptive immunity in patients with GBM via a T1IFN-based trajectory and identified a gene panel signature of TTFields effects on T cell activation and clonal expansion. Collectively, these studies defined a therapeutic strategy using TTFields as cancer immunotherapy in GBM and potentially other solid tumors.

Chemoresistance of glioblastoma (GBM) is a feature of this devastating disease. This study is to determine the relationship between Hedgehog (HH)/Gli1 signaling pathway and chemoresistance to temozolomide (TMZ) in human GBM. We analyzed Gli1 nuclear staining and O6-methylguanine DNA methyltransferase (MGMT) expression in 48 cases of primary GBM tissues by immunohistochemistry. Quantitative PCR, western blot, methylation-specific PCR, cell proliferation and apoptosis assay were used to investigate changes of MGMT expression and chemosensitivity to TMZ after manipulating HH/Gli1 signaling activity in A172 and U251 GBM cell lines. Chromatin immunoprecipitation assay was utilized to identify potential Gli1 potential binding sites in MGMT gene promoter region. We established GBM xenografts using U251 cells to assess whether inhibiting HH/Gli1 signaling activity restored chemosensitivity to TMZ. O6-Methylguanine DNA methyltransferase-positive GBM tissues had a significantly higher rate of Gli1 nuclear staining than MGMT-negative ones (67.7% vs. 32.3%, p = 0.0159). Activation of HH/Gli1 signaling by pcDNA3.1-Gli1 cell transfection in A172 cells led to increased MGMT expression and enhanced resistance to TMZ treatment. Inhibition of the HH/Gli1 signaling by cyclopamine in U251 cells resulted in decreased MGMT expression and increased sensitivity to TMZ treatment. Both ways altered MGMT levels without changing the MGMT promoter methylation. The potential binding site of Gli1 in the MGMT gene promoter region was located at – 411 to − 403 bp upstream the transcriptional start site. The in vivo study revealed a synergistic effect on tumor growth inhibition with the combined administration of cyclopamine and TMZ. This study shows that HH/Gli1 signaling pathway regulates MGMT expression and chemoresistance to TMZ in human GBM independent from MGMT promoter methylation status, which offers a potential target to restore chemosensitivity to TMZ in a fraction of GBM with high MGMT expression.

Recurrent glioblastoma (rGBM) is a highly aggressive form of brain cancer that poses a significant challenge for treatment in neuro-oncology, and the survival status of patients after relapse usually means rapid deterioration, thus becoming the leading cause of death among patients. In recent years, immunotherapy has emerged as a promising strategy for the treatment of recurrent glioblastoma by stimulating the body’s immune system to recognize and attack cancer cells, which could be used in combination with other treatments such as surgery, radiation, and chemotherapy to improve outcomes for patients with recurrent glioblastoma. This therapy combines several key methods such as the use of monoclonal antibodies, chimeric antigen receptor T cell (CAR-T) therapy, checkpoint inhibitors, oncolytic viral therapy cancer vaccines, and combination strategies. In this review, we mainly document the latest immunotherapies for the treatment of glioblastoma and especially focus on rGBM.

Immunotherapy has limited efficacy in glioblastoma (GBM) due to the blood–brain barrier and the immunosuppressed or “cold” tumor microenvironment (TME) of GBM, which is dominated by immune-inhibitory cells and depleted of CTL and dendritic cells (DC). Here, we report the development and application of a machine learning precision method to identify cell fate determinants (CFD) that specifically reprogram GBM cells into induced antigen-presenting cells with DC-like functions (iDC-APC). In murine GBM models, iDC-APCs acquired DC-like morphology, regulatory gene expression profile, and functions comparable to natural DCs. Among these acquired functions were phagocytosis, direct presentation of endogenous antigens, and cross-presentation of exogenous antigens. The latter endowed the iDC-APCs with the ability to prime naïve CD8+ CTLs, a hallmark DC function critical for antitumor immunity. Intratumor iDC-APCs reduced tumor growth and improved survival only in immunocompetent animals, which coincided with extensive infiltration of CD4+ T cells and activated CD8+ CTLs in the TME. The reactivated TME synergized with an intratumor soluble PD1 decoy immunotherapy and a DC-based GBM vaccine, resulting in robust killing of highly resistant GBM cells by tumor-specific CD8+ CTLs and significantly extended survival. Lastly, we defined a unique CFD combination specifically for the human GBM to iDC-APC conversion of both glioma stem-like cells and non–stem-like cell GBM cells, confirming the clinical utility of a computationally directed, tumor-specific conversion immunotherapy for GBM and potentially other solid tumors.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment (TME) and dampen the immune response, negatively affecting patient survival. Therefore, targeting TAMs could address the limitations of current cancer treatments. However, drug development in this area remains limited. The leukocyte-associated Ig-like receptor 1 (LAIR1), also called CD305, is prominently expressed on the surface of TAMs. We have uncovered what we believe to be a previously unrecognized immunosuppressive LAIR1/factor XIII A/collagen IV pathway across various cancer types. Inhibition of LAIR1, either through knockout ( Lair1 ^–/– ), antibody blockade (anti-Lair1 antibody), or a chimeric antigen receptor (CAR) design (3-in-1 CAR by combining tumor targeting, T cell trafficking, and remodeling of the immunosuppressive TME in 1 CAR construct) provided an enhanced antitumor response. LAIR1 inhibition enhanced peripheral and intratumoral CD8 memory T cell populations, induced a phenotypic shift of M2-like macrophages toward M1 macrophages, and normalized tumor collagen IV and structural components in the TME, facilitating effective tumor–T cell interactions and tumor suppression. Enhanced antitumor responses were observed when Lair1 ^–/– or anti-Lair1 antibody was used alone or in combination with CAR T cells or when the 3-in-1 CAR T cells were used solely in tumor models resistant to chemotherapy–radiation–programmed cell death protein 1 (PD-1) blockade. These findings position LAIR1 inhibition as a promising strategy for cancer immunotherapies.

Tumor treating fields (TTFields) use low-intensity, alternating electric fields to exert antitumor activity and have demonstrated efficacy against multiple cancers, including glioblastoma (GBM). Unfortunately, cancer cells inevitably develop resistance to TTFields, highlighting the need to elucidate the underlying mechanisms to develop approaches to induce durable responses. Using a gene network–based machine learning algorithm, we interrogated TTFields-resistant GBM cells and uncovered a regulatory axis anchored by the prostaglandin E2 receptor 3 (EP3) and the transcription factor zinc finger 488 (ZNF488). Mechanistically, TTFields induced EP3 upregulation and nuclear envelope localization, where it formed a complex with ZNF488 to induce resistance to TTFields by promoting self-renewal of glioma stem-like cells (GSC). Overexpression of EP3 and/or ZNF488 in TTFields-sensitive GSC conferred resistance and enhanced self-renewal, whereas expression of noninteracting mutants of these proteins abrogated the formation of the nuclear complex and prevented resistance. Inhibition of either partner in this protein complex in resistant GSC, including those freshly isolated from TTFields-resistant GBM tumors, resensitized cells to the cytotoxic effects of TTFields, concomitant with reduced self-renewal and in vivo tumorigenicity. Importantly, inhibition of EP3 in TTFields-sensitive GSC preemptively halted the development of resistance. The EP3–ZNF488 axis was significantly upregulated in TTFields-resistant GBM tumors, and coexpression of EP3 and ZNF488 in other cancers correlated with lower survival rates. Collectively, these results indicate that the nuclear EP3–ZNF488 axis is necessary and sufficient to establish TTFields resistance, underscoring the potential to target this axis to prevent or reverse resistance in GBM and possibly other cancers. Significance: The EP3–ZNF488 master regulatory axis in cancer stem-like cells drives resistance to treatments like tumor treating fields, opening avenues for developing strategies to enhance therapeutic efficacy.