Glioblastoma multiforme (GBM) modulates the immune system to engance its malignant potential. Signal transducer and activator of transcription 3 (STAT3) activation is a regulatory node in modulating ...the immune microenvironment in several human tumors, including GBM. To investigate whether STAT3 inhibition might enhance anti-tumor responses, we inhibited STAT3 signaling using small interfering RNA against STAT3. We tested the human GBM cell lines U87, U251, and HS683, which are known to constitutively express high levels of phospho-STAT3. STAT3 inhibition resulted in enhanced expression of several pro-inflammatory cytokines and chemokines and supernatants from STAT3-silenced human GBM cell lines increased lipopolysaccharide-induced dendritic cell activation in vitro. We obtained comparable results when STAT3 activity was suppressed with specific small molecule inhibitors. Our results support the hypothesis that activated STAT3 contributes to the immunosuppressive microenvironment in GBM and support previous studies implicating STAT3 as a potential target for immunotherapy.
Microglia and macrophages, two myeloid cell lineages with different origins, make up the majority of immune cells present in glioblastoma (GBM). However, much of the literature does not distinguish ...between microglia and macrophages, despite a growing body of evidence that demonstrates key structural and functional differences between the cell types. Furthermore, the current M1/M2 paradigm used to sub-classify microglia and macrophages has proven to be incomplete at best, with the growing amount of in vivo and genomic data incompatible with this dichotomy. Finally, a number of studies have already established that in the setting of the GBM tumor microenvironment, both microglia and macrophages are complicit in tumor progression. This review highlights the differences between microglia and macrophages, particularly in the context of GBM, and discusses at length several potential therapeutic strategies made possible by understanding specific pro-tumor and anti-tumor pathways in these myeloid populations. Ultimately, investigating the differences between microglia and macrophages offers insight into the progression of GBM, its marked resistance to current immunotherapy regimens, and future directions for new treatment modalities.
The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the ...standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity.
Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles ...(CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.
Tumor mutation burden is an imperfect proxy of tumor foreignness and has therefore failed to consistently demonstrate clinical utility in predicting responses in the context of immunotherapy. We ...evaluated mutations in regions of the genome that are unlikely to undergo loss in a pan-cancer analysis across 31 tumor types (n = 9,242) and eight immunotherapy-treated cohorts of patients with non-small-cell lung cancer, melanoma, mesothelioma, and head and neck cancer (n = 524). We discovered that mutations in single-copy regions and those present in multiple copies per cell constitute a persistent tumor mutation burden (pTMB) which is linked with therapeutic response to immune checkpoint blockade. Persistent mutations were retained in the context of tumor evolution under selective pressure of immunotherapy and tumors with a high pTMB content were characterized by a more inflamed tumor microenvironment. pTMB imposes an evolutionary bottleneck that cancer cells cannot overcome and may thus drive sustained immunologic tumor control in the context of immunotherapy.
Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune ...checkpoint inhibitors including anti‐PD‐1 or CTLA4. We here investigate the expression and efficacy of a novel immune‐checkpoint inhibitor, called LAG‐3. We show that LAG‐3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG‐3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG‐3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG‐3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG‐3 in the treatment of glioblastoma.
What's new?
Glioblastoma derives some of its lethality from its ability to escape destruction by the immune system. Researchers have begun investigating immune checkpoint inhibitors as a tool to combat glioblastoma. Here, the authors report on a novel immune‐checkpoint inhibitor, LAG‐3. In a mouse model of glioblastoma, they successfully improved survival by eliminating LAG‐3, either by genetic knockout or using antibodies against it. They show that TILs from human glioblastoma samples express LAG‐3, and that high LAG‐3 expression correlates with reduced interferon release. The authors propose that anti‐LAG‐3, alone or in combination with other anti‐PD‐1 treatment, could improve glioblastoma treatment.
Patients with small-cell lung cancer (SCLC) have an exceptionally poor prognosis, calling for improved real-time noninvasive biomarkers of therapeutic response.
We performed targeted error-correction ...sequencing on 171 serial plasmas and matched white blood cell (WBC) DNA from 33 patients with metastatic SCLC who received treatment with chemotherapy (n = 16) or immunotherapy-containing (n = 17) regimens. Tumor-derived sequence alterations and plasma aneuploidy were evaluated serially and combined to assess changes in total cell-free tumor load (cfTL). Longitudinal dynamic changes in cfTL were monitored to determine circulating cell-free tumor DNA (ctDNA) molecular response during therapy.
Combined tiered analyses of tumor-derived sequence alterations and plasma aneuploidy allowed for the assessment of ctDNA molecular response in all patients. Patients classified as molecular responders (n = 9) displayed sustained elimination of cfTL to undetectable levels. For 14 patients, we observed initial molecular responses, followed by ctDNA recrudescence. A subset of patients (n = 10) displayed a clear pattern of molecular progression, with persistence of cfTL across all time points. Molecular responses captured the therapeutic effect and long-term clinical outcomes in a more accurate and rapid manner compared with radiographic imaging. Patients with sustained molecular responses had longer overall (log-rank P = 0.0006) and progression-free (log-rank P < 0.0001) survival, with molecular responses detected on average 4 weeks earlier than imaging.
ctDNA analyses provide a precise approach for the assessment of early on-therapy molecular responses and have important implications for the management of patients with SCLC, including the development of improved strategies for real-time tumor burden monitoring. See related commentary by Pellini and Chaudhuri, p. 2176.
The cone-beam computed tomography (CBCT)-guided small animal radiation research platform (SARRP) has provided unique opportunities to test radiobiologic hypotheses. However, CBCT is less adept to ...localize soft tissue targets growing in a low imaging contrast environment. Three-dimensional bioluminescence tomography (BLT) provides strong image contrast and thus offers an attractive solution. We introduced a novel and efficient BLT-guided conformal radiation therapy and demonstrated it in an orthotopic glioblastoma (GBM) model.
A multispectral BLT system was integrated with SARRP for radiation therapy (RT) guidance. GBM growth curve was first established by contrast CBCT/magnetic resonance imaging (MRI) to derive equivalent sphere as approximated gross target volume (aGTV). For BLT, mice were subject to multispectral bioluminescence imaging, followed by SARRP CBCT imaging and optical reconstruction. The CBCT image was acquired to generate anatomic mesh for the reconstruction and RT planning. To ensure high accuracy of the BLT-reconstructed center of mass (CoM) for target localization, we optimized the optical absorption coefficients μ
by minimizing the distance between the CoMs of BLT reconstruction and contrast CBCT/MRI-delineated GBM volume. The aGTV combined with the uncertainties of BLT CoM localization and target volume determination was used to generate estimated target volume (ETV). For conformal irradiation procedure, the GBM was first localized by the predetermined ETV centered at BLT-reconstructed CoM, followed by SARRP radiation. The irradiation accuracy was qualitatively confirmed by pathologic staining.
Deviation between CoMs of BLT reconstruction and contrast CBCT/MRI-imaged GBM is approximately 1 mm. Our derived ETV centered at BLT-reconstructed CoM covers >95% of the tumor volume. Using the second-week GBM as an example, the ETV-based BLT-guided irradiation can cover 95.4% ± 4.7% tumor volume at prescribed dose. The pathologic staining demonstrated the BLT-guided irradiated area overlapped well with the GBM location.
The BLT-guided RT enables 3-dimensional conformal radiation for important orthotopic tumor models, which provides investigators a new preclinical research capability.
Abstract
Introduction: Radiation therapy (RT) has the potential to elicit a systemic immune response beyond the field of radiation (abscopal effect), which can be enhanced by immune checkpoint ...blockade (ICB) leading to improved anti-tumor immune responses. Immune cell repertoire dynamics during combined immunotherapy and radiation are not well understood.
Methods: We performed whole exome sequencing (WES) and T cell receptor (TCR) sequencing utilizing serial tumor biopsies and peripheral blood samples from 72 patients with advanced non-small cell lung cancer (NSCLC) treated on a phase 2 clinical trial of stereotactic body RT (SBRT) and pembrolizumab (NCT02492568). Best overall response was evaluated by RECIST criteria at 12 weeks on-therapy. Serial biopsies from a non-irradiated tumor site and peripheral blood samples were obtained prior to therapy and after SBRT and/or two cycles of pembrolizumab. 58 matched tumor/normal DNA pairs were analyzed by WES and 120 baseline/on-therapy tumor and peripheral blood mononuclear cell (PBMC) samples were analyzed by TCR-β CDR3 next generation sequencing. We performed differential intratumoral TCR clonotypic abundance analyses and examined the representation and dynamics of these clones in peripheral blood. The most expanded and regressed TCR clonotypes, corresponding to fold changes in productive frequency of TCR clones with an FDR<0.05 were selected to determine intratumoral and peripheral T cell repertoire reshaping during therapy.
Results: Tumor mutation burden was not significantly correlated with response to combined immuno-radiation therapy (P = 0.16). Paired TCR-β CDR3 sequencing of baseline and on-treatment tumors revealed a significantly higher intratumoral TCR density in the on-treatment tumors after combined SBRT and pembrolizumab compared to pembrolizumab alone (P= 0.013). TCR clonotypic differential abundance analysis revealed a significantly higher fraction of expanding and regressing clones that were shared between the tumor and peripheral blood compartments after combination therapy (P= 0.045). Combination therapy induced reshaping of the peripheral TCR repertoire seen in significant TCR clonotypic expansions compared to pembrolizumab alone (P=0.0078). In all 3 patients with a complete response after combination therapy, a distinct pattern of TCR repertoire reshaping in the TIL and peripheral blood compartments was observed despite absent or <50% tumor PD-L1 expression. While intratumoral TCR dynamic changes were appreciated in patients achieving a clinical response independent of therapy, reshaping of the peripheral TCR repertoire was more prominent in responders in the radiation therapy group.
Conclusion: Combined radiation with ICB induces unique dynamic changes in the peripheral and intratumoral T cell repertoire that may be reflective of an abscopal effect and linked with clinical outcomes.
Citation Format: Zineb Belcaid, Willemijn S. Theelen, James R. White, Ashok Sivakumar, Archana Balan, Noushin Niknafs, Leonardo Ferreira, Vilmos Adleff, Victor E. Velculescu, Paul Baas, Rachel Karchin, Valsamo Anagnostou. Immunogenomic mechanisms of response and resistance to combined radiation and immunotherapy in lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1662.
Immune checkpoint inhibitors such as anti-programmed cell death protein 1 (anti-PD-1) have shown promise for the treatment of cancers such as melanoma, but results for glioblastoma (GBM) have been ...disappointing thus far. It has been suggested that GBM has multiple mechanisms of immunosuppression, indicating a need for combinatorial treatment strategies. It is well understood that GBM increases glutamate in the tumor microenvironment (TME); however, the significance of this is not well understood. The authors posit that glutamate upregulation in the GBM TME is immunosuppressive. The authors utilized a novel glutamate modulator, BHV-4157, to determine synergy between glutamate modulation and the well-established anti-PD-1 immunotherapy for GBM.
C57BL/6J mice were intracranially implanted with luciferase-tagged GL261 glioma cells. Mice were randomly assigned to the control, anti-PD-1, BHV-4157, or combination anti-PD-1 plus BHV-4157 treatment arms, and median overall survival was assessed. In vivo microdialysis was performed at the tumor site with administration of BHV-4157. Intratumoral immune cell populations were characterized with immunofluorescence and flow cytometry.
The BHV-4157 treatment arm demonstrated improved survival compared with the control arm (p < 0.0001). Microdialysis demonstrated that glutamate concentration in TME significantly decreased after BHV-4157 administration. Immunofluorescence and flow cytometry demonstrated increased CD4+ T cells and decreased Foxp3+ T cells in mice that received BHV-4157 treatment. No survival benefit was observed when CD4+ or CD8+ T cells were depleted in mice prior to BHV-4157 administration (p < 0.05).
In this study, the authors showed synergy between anti-PD-1 immunotherapy and glutamate modulation. The authors provide a possible mechanism for this synergistic benefit by showing that BHV-4157 relies on CD4+ and CD8+ T cells. This study sheds light on the role of excess glutamate in GBM and provides a basis for further exploring combinatorial approaches for the treatment of this disease.
PDF
