Next-generation agents for fluorescence-guided glioblastoma surgery.

Glioblastoma is a fast-growing and aggressive form of brain cancer. Even with maximal treatment, patients show a low median survival and are often subjected to a high recurrence incidence. The currently available treatments require multimodal management, including maximal safe surgical resection, followed by radiation and chemotherapy. Because of the infiltrative glioblastoma nature, intraoperative differentiation of cancer tissue from normal brain parenchyma is very challenging, and this accounts for the low rate of complete tumor resection. For these reasons, clinicians have increasingly used various intraoperative adjuncts to improve surgical results, such as fluorescent agents. However, most of the existing fluorophores show several limitations such as poor selectivity, photostability, photosensitization and high costs. This could limit their application to successfully improve glioblastoma resection. In the present perspective, we highlight the possibility to develop next-generation fluorescent tools able to more selectively label cancer cells during surgical resection.

Response assessment of GBM during immunotherapy by delayed contrast treatment response assessment maps.

Introduction: Assessing the treatment response of glioblastoma multiforme during immunotherapy (IT) is an open issue. Treatment response assessment maps (TRAMs) might help distinguish true tumor progression (TTP) and pseudoprogression (PsP) in this setting. Methods: We recruited 16 naïve glioblastoma patients enrolled in a phase II trial consisting of the Stupp protocol (a standardized treatment for glioblastoma involving combined radiotherapy and chemotherapy with temozolomide, followed by adjuvant temozolomide) plus IT with dendritic cells. Patients were followed up till progression or death; seven underwent a second surgery for suspected progression. Clinical, immunological, and MRI data were collected from all patients and histology in case of second surgery. Patients were classified as responders (progression-free survival, PFS > 12 months), and non-responders (PFS ≤ 12), HIGH-NK (natural killer cells, i.e., immunological responders), and LOW-NK (immunological non-responders) based on immune cell counts in peripheral blood. TRAMs differentiate contrast-enhancing lesions with different washout dynamics into hypothesized tumoral (conventionally blue-colored) vs. treatment-related (red-colored). Results: Using receiver operating characteristic (ROC) curves, a threshold of -0.066 in VBlue/VCE (volume of the blue portion of tumoral area/volume of contrast enhancement) variation between values obtained in the MRI performed before PsP/TTP and at TTP/PSP allowed to discriminate TTP from PsP with a sensitivity of 71.4% and a specificity of 100%. Among HIGH-NK patients, at month 6 there was a significant reduction compared to baseline and month 2 in median "blue" volumes. Discussion: In conclusion, in our pilot study TRAMs support the discrimination between tumoral and treatment-related enhancing features in immunological responders vs. non-responders, the distinction between PsP and TTP, and might provide surrogate markers of immunological response.

The Immunomodulatory Effects of Fluorescein-Mediated Sonodynamic Treatment Lead to Systemic and Intratumoral Depletion of Myeloid-Derived Suppressor Cells in a Preclinical Malignant Glioma Model.

Fluorescein-mediated sonodynamic therapy (FL-SDT) is an extremely promising approach for glioma treatment, resulting from the combination of low-intensity focused ultrasound (FUS) with a sonosensitizer. In the present study, we evaluated the efficacy and immunomodulation of SDT with fluorescein as the sonosensitizer in immunocompetent GL261 glioma mice for the first time. In vitro studies demonstrated that the exposure of GL261 cells to FL-SDT induced immunogenic cell death and relevant upregulation of MHC class I, CD80 and CD86 expression. In vivo studies were then performed to treat GL261 glioma-bearing mice with FL-SDT, fluorescein alone, or FUS alone. Perturbation of the glioma-associated macrophage subset within the immune microenvironment was induced by all the treatments. Notably, a relevant depletion of myeloid-derived suppressor cells (MDSCs) and concomitant robust infiltration of CD8+ T cells were observed in the SDT-FL-treated mice, resulting in a significant radiological delay in glioma progression and a consequent improvement in survival. Tumor control and improved survival were also observed in mice treated with FL alone (median survival 41.5 days, p > 0.0001 compared to untreated mice), reflecting considerable modulation of the immune microenvironment. Interestingly, a high circulating lymphocyte-to-monocyte ratio and a very low proportion of MDSCs were predictive of better survival in FL- and FL-SDT-treated mice than in untreated and FUS-treated mice, in which elevated monocyte and MDSC frequencies correlated with worse survival. The immunostimulatory potential of FL-SDT treatment and the profound modulation of most immunosuppressive components within the microenvironment encouraged the exploration of the combination of FL-SDT with immunotherapeutic strategies.

Coexisting cancer stem cells with heterogeneous gene amplifications, transcriptional profiles, and malignancy are isolated from single glioblastomas.

Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

Deciphering the Labyrinthine System of the Immune Microenvironment in Recurrent Glioblastoma: Recent Original Advances and Lessons from Clinical Immunotherapeutic Approaches.

The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.

Revisiting the Immunological Aspects of Temozolomide Considering the Genetic Landscape and the Immune Microenvironment Composition of Glioblastoma.

The microenvironment (ME) plays a critical role in causing glioblastoma (GBM) to be a moving and incurable target. The main features governing the interaction between cancer cells and the ME include dependency, promotion, and in rare cases, even competition. In the original Stupp protocol, the alkylating agent temozolomide (TMZ) is the first-line chemotherapy drug to treat GBM, and it is broadly used together or after radiotherapy. Some studies have described TMZ as an adjuvant to other therapeutic approaches including immunotherapy because of its ability to induce an immunogenic death of cancer cells. TMZ also exerts immunomodulatory effects on the tumor and immune ME. These findings support the coexistence of two circuits, i.e., one that subverts local immunosuppressive mechanisms and another that exerts a harmful influence on the peripheral immune response. A bias toward the latter can drive the failure of treatments based on the combination of chemotherapy and immunotherapy approaches. In this review, we will reanalyze how intrinsic and acquired resistance to TMZ impacts the immunomodulatory effects previously described by way of inducing a functional alteration of local immune cells and promoting immunosuppression and how different components of the immune ME, with particular attention to tumor-associated macrophages and microglia, can cause TMZ resistance to circumvent potential local immunogenic mechanisms.

ERBB3 overexpression due to miR-205 inactivation confers sensitivity to FGF, metabolic activation, and liability to ERBB3 targeting in glioblastoma.

In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.

Sonodynamic Therapy for the Treatment of Intracranial Gliomas.

High-grade gliomas are the most common and aggressive malignant primary brain tumors. Current therapeutic schemes include a combination of surgical resection, radiotherapy and chemotherapy; even if major advances have been achieved in Progression Free Survival and Overall Survival for patients harboring high-grade gliomas, prognosis still remains poor; hence, new therapeutic options for malignant gliomas are currently researched. Sonodynamic Therapy (SDT) has proven to be a promising treatment combining the effects of low-intensity ultrasound waves with various sound-sensitive compounds, whose activation leads to increased immunogenicity of tumor cells, increased apoptotic rates and decreased angiogenetic potential. In addition, this therapeutic technique only exerts its cytotoxic effects on tumor cells, while both ultrasound waves and sensitizing compound are non-toxic per se. This review summarizes the present knowledge regarding mechanisms of action of SDT and currently available sonosensitizers and focuses on the preclinical and clinical studies that have investigated its efficacy on malignant gliomas. To date, preclinical studies implying various sonosensitizers and different treatment protocols all seem to confirm the anti-tumoral properties of SDT, while first clinical trials will soon start recruiting patients. Accordingly, it is crucial to conduct further investigations regarding the clinical applications of SDT as a therapeutic option in the management of intracranial gliomas.

Altered Metabolism in Glioblastoma: Myeloid-Derived Suppressor Cell (MDSC) Fitness and Tumor-Infiltrating Lymphocyte (TIL) Dysfunction.

The metabolism of glioblastoma (GBM), the most aggressive and lethal primary brain tumor, is flexible and adaptable to different adverse conditions, such as nutrient deprivation. Beyond glycolysis, altered lipid metabolism is implicated in GBM progression. Indeed, metabolic subtypes were recently identified based on divergent glucose and lipid metabolism. GBM is also characterized by an immunosuppressive microenvironment in which myeloid-derived suppressor cells (MDSCs) are a powerful ally of tumor cells. Increasing evidence supports the interconnection between GBM and MDSC metabolic pathways. GBM cells exert a crucial contribution to MDSC recruitment and maturation within the tumor microenvironment, where the needs of tumor-infiltrating lymphocytes (TILs) with antitumor function are completely neglected. In this review, we will discuss the unique or alternative source of energy exploited by GBM and MDSCs, exploring how deprivation of specific nutrients and accumulation of toxic byproducts can induce T-cell dysfunction. Understanding the metabolic programs of these cell components and how they impact fitness or dysfunction will be useful to improve treatment modalities, including immunotherapeutic strategies.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

MR-Spectroscopy and Survival in Mice with High Grade Glioma Undergoing Unrestricted Ketogenic Diet.

Glioblastoma multiforme (GBM) is considered the most malignant form of primary brain tumor. Despite multimodal treatment, prognosis remains poor. Ketogenic diet (KD) has been suggested for the treatment of GBM. In this study, the syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on the metabolic responses of the tumor using 7T magnetic resonance imaging/spectroscopy. GL261 cells were injected into the caudate nucleus of mice. Following implantation, animals were fed with standard chow or underwent a KD. 18 days after initiating the diet, mice fed with KD displayed significantly higher plasmatic levels of ketone bodies and survived longer than those fed with the standard diet. Decreased concentrations of gamma-aminobutyric acid, N-Acetyl-Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days into the KD, while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The accumulation of bHB in the tumor tissue in mice undergoing the KD, may suggest either elevated uptake/release of bHB by tumor cells, or the inability of tumor cells in this context to use it for mitochondrial metabolism.

High tumor mutational burden and T-cell activation are associated with long-term response to anti-PD1 therapy in Lynch syndrome recurrent glioblastoma patient.

BACKGROUND: Glioblastomas (GBMs) in patients harboring somatic or germinal mutations of mismatch-repair (MMR) genes exhibit a hypermutable phenotype. Here, we describe a GBM patient with increased tumor mutational burden and germline MMR mutations, treated using anti-PD1 therapy. METHODS: A woman with newly diagnosed GBM (nGBM) was treated by surgery, radiotherapy, and temozolomide. The tumor recurred after 13 months leading to a second surgery and treatment with nivolumab. Whole-exome sequencing was performed on the nGBM, recurrent GBM (rGBM), and blood. Immune infiltration was investigated by immunohistochemistry and the immune response in the blood during treatment was analyzed by flow cytometry. RESULTS: High density of infiltrating CD163 + cells was found in both GBM specimens. Large numbers of CD3 + and CD8 + T cells were homogeneously distributed in the nGBM. The infiltration of CD4 + T cells and a different CD8 + T cell density were observed in the rGBM. Both GBM shared 12,431 somatic mutations, with 113 substitutions specific to the nGBM and 1,683 specific to the rGBM. Germline variants included pathogenic mutation in the MSH2 (R359S) gene, suggesting the diagnosis of Lynch syndrome. Systemic immunophenotyping revealed the generation of CD8 + T memory cells and persistent activation of CD4 + T cells. The patient is still receiving nivolumab 68 months after the second surgery. CONCLUSIONS: Our observations indicate that the hypermutator phenotype associated with germinal mutations of MMR genes and abundant T-cell infiltration contributes to a durable clinical benefit sustained by a persistent and robust immune response during anti-PD1 therapy.

PGE2 Is Crucial for the Generation of FAST Whole- Tumor-Antigens Loaded Dendritic Cells Suitable for Immunotherapy in Glioblastoma.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5-7 days of differentiation with GM-CSF and IL-4 followed by 2-3 days of activation/maturation. In attempts to shorten the vaccine's production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE2 in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.

Advanced MRI Assessment during Dendritic Cell Immunotherapy Added to Standard Treatment Against Glioblastoma.

Evaluating changes induced by immunotherapies (IT) on conventional magnetic resonance imaging (MRI) is difficult because those treatments may produce inflammatory responses. To explore the potential contribution of advanced MRI to distinguish pseudoprogression (PsP) and true tumor progression (TTP), and to identify patients obtaining therapeutic benefit from IT, we examined aMRI findings in newly diagnosed glioblastoma treated with dendritic cell IT added to standard treatment. We analyzed longitudinal MRIs obtained in 22 patients enrolled in the EUDRACT N° 2008-005035-15 trial. According to RANO criteria, we observed 18 TTP and 8 PsP. Comparing MRI performed at the time of TTP/PsP with the previous exam performed two months before, a difference in cerebral blood volume ΔrCBVmax ≥ 0.47 distinguished TTP from PsP with a sensitivity of 67% and specificity of 75% (p = 0.004). A decrease in minimal apparent diffusion coefficient rADCmin (1.15 vs. 1.01, p = 0.003) was observed after four vaccinations only in patients with a persistent increase of natural killer cells (response effectors during IT) in peripheral blood. Basal rADCmin > 1 was independent predictor of longer progression free (16.1 vs. 9 months, p = 0.0001) and overall survival (32.8 vs. 17.5 months, p = 0.0005). In conclusion, rADC predicted response to immunotherapy and survival; Apparent Diffusion Coefficient (ADC) and Cerebral Blood Volume (CBV) modifications over time help differentiating PsP from TTP at onset.

ABCC3 Expressed by CD56dim CD16+ NK Cells Predicts Response in Glioblastoma Patients Treated with Combined Chemotherapy and Dendritic Cell Immunotherapy.

Recently, we found that temozolomide (TMZ) can upregulate the expression of the multidrug-resistance protein ABCC3 in NK cells from both glioma-bearing mice and glioblastoma patients treated with dendritic cell immunotherapy combined with TMZ, allowing NK cells to escape apoptosis and favoring their role as antitumor effector cells. Here, we demonstrate that CD56dim NK cells expressing CD16+ are predominant in patients surviving more than 12 months after surgery without disease progression. CD56dim CD16+ NK cells co-expressed high levels of ABCC3 and IFN-. Notably, not only basal but also TMZ-induced ABCC3 expression was related to a strong, long-term NK cell response and a better prognosis of patients. The identification of the single nucleotide polymorphism (SNP) rs35467079 with the deletion of a cytosine (-897DelC) in the promoter region of the ABCC3 gene resulted associated with a better patient outcome. ABCC3 expression in patients carrying DelC compared to patients with reference haplotype was higher and modulated by TMZ. The transcription factor NRF2, involved in ABCC3 induction, was phosphorylated in CD56dim CD16+ NK cells expressing ABCC3 under TMZ treatment. Thus, ABCC3 protein and the SNP -897DelC can play a predictive role in patients affected by GBM, and possibly other cancers, treated with dendritic cell immunotherapy combined with chemotherapy.

B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.

BACKGROUND: The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM. METHODS: We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. FINDINGS: B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines. INTERPRETATION: We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. FUND: Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.

BACKGROUND: The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). METHODS: In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. RESULTS: Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. CONCLUSIONS: TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.