Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.

BACKGROUND: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo. METHODS: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors. RESULTS: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors. CONCLUSIONS: By turning "two keys" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.

Corrigendum: Myelomodulatory treatments augment the therapeutic benefit of oncolytic viroimmunotherapy in murine models of malignant peripheral nerve sheath tumors.

[This corrects the article DOI: 10.3389/fimmu.2024.1384623.].

Myelomodulatory treatments augment the therapeutic benefit of oncolytic viroimmunotherapy in murine models of malignant peripheral nerve sheath tumors.

Introduction: Malignant peripheral nerve sheath tumors (MPNST) pose a significant therapeutic challenge due to high recurrence rates after surgical resection and a largely ineffective response to traditional chemotherapy. An alternative treatment strategy is oncolytic viroimmunotherapy, which can elicit a durable and systemic antitumor immune response and is Food and Drug Administration (FDA)-approved for the treatment of melanoma. Unfortunately, only a subset of patients responds completely, underscoring the need to address barriers hindering viroimmunotherapy effectiveness. Methods: Here we investigated the therapeutic utility of targeting key components of the MPNST immunosuppressive microenvironment to enhance viroimmunotherapy's antitumor efficacy in three murine models, one of which showed more immunogenic characteristics than the others. Results: Myelomodulatory therapy with pexidartinib, a small molecule inhibitor of CSF1R tyrosine kinase, and the oncolytic herpes simplex virus T-VEC exhibited the most significant increase in median survival time in the highly immunogenic model. Additionally, targeting myeloid cells with the myelomodulatory therapy trabectedin, a small molecule activator of caspase-8 dependent apoptosis, augmented the survival benefit of T-VEC in a less immunogenic MPNST model. However, tumor regressions or shrinkages were not observed. Depletion experiments confirmed that the enhanced survival benefit relied on a T cell response. Furthermore, flow cytometry analysis following combination viroimmunotherapy revealed decreased M2 macrophages and myeloid-derived suppressor cells and increased tumor-specific gp70+ CD8 T cells within the tumor microenvironment. Discussion: In summary, our findings provide compelling evidence for the potential to leverage viroimmunotherapy with myeloid cell targeting against MPNST and warrant further investigation.

Disease Control and Toxicity Outcomes after Stereotactic Ablative Radiation Therapy for Recurrent and/or Metastatic Cancers in Young-Adult and Pediatric Patients.

BACKGROUND: Pediatric patients with metastatic and/or recurrent solid tumors have poor survival outcomes despite standard-of-care systemic therapy. Stereotactic ablative radiation therapy (SABR) may improve tumor control. We report the outcomes with the use of SABR in our pediatric solid tumor population. METHODS: This was a single-institutional study in patients < 30 years treated with SABR. The primary endpoint was local control (LC), while the secondary endpoints were progression-free survival (PFS), overall survival (OS), and toxicity. The survival analysis was performed using Kaplan-Meier estimates in R v4.2.3. RESULTS: In total, 48 patients receiving 135 SABR courses were included. The median age was 15.6 years (interquartile range, IQR 14-23 y) and the median follow-up was 18.1 months (IQR: 7.7-29.1). The median SABR dose was 30 Gy (IQR 25-35 Gy). The most common primary histologies were Ewing sarcoma (25%), rhabdomyosarcoma (17%), osteosarcoma (13%), and central nervous system (CNS) gliomas (13%). Furthermore, 57% of patients had oligometastatic disease (≤5 lesions) at the time of SABR. The one-year LC, PFS, and OS rates were 94%, 22%, and 70%, respectively. No grade 4 or higher toxicities were observed, while the rates of any grade 1, 2, and 3 toxicities were 11.8%, 3.7%, and 4.4%, respectively. Patients with oligometastatic disease, lung, or brain metastases and those who underwent surgery for a metastatic site had a significantly longer PFS. LC at 1-year was significantly higher for patients with a sarcoma histology (95.7% vs. 86.5%, p = 0.01) and for those who received a biological equivalent dose (BED10) > 48 Gy (100% vs. 91.2%, p = 0.001). CONCLUSIONS: SABR is well tolerated in pediatric patients with 1-year local failure and OS rates of <10% and 70%, respectively. Future studies evaluating SABR in combination with systemic therapy are needed to address progression outside of the irradiated field.

Enhanced IL-12 transgene expression improves oncolytic viroimmunotherapy.

Introduction: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with unacceptably low cure rates occurring often in patients with neurofibromatosis 1 defects. To investigate oncolytic Herpes Simplex Virus (oHSV) as an immunotherapeutic approach, we compared viral replication, functional activity, and immune response between unarmed and interleukin 12 (IL-12)-armed oncolytic viruses in virus-permissive (B109) and -resistant (67C-4) murine MPNSTs. Methods: This study compared two attenuated IL-12-oHSVs with γ134.5 gene deletions (Δγ134.5) and the same transgene expression cassette. The primary difference in the IL-12-oHSVs was in their ability to counter the translational arrest response in infected cells. Unlike M002 (Δγ134.5, mIL-12), C002 (Δγ134.5, mIL-12, IRS1) expresses an HCMV IRS1 gene and evades dsRNA activated translational arrest in infected cells. Results and discussion: Our results show that oHSV replication and gene expression results in vitro were not predictive of oHSV direct oncolytic activity in vivo. Tumors that supported viral replication in cell culture studies resisted viral replication by both oHSVs and restricted M002 transgene expression in vivo. Furthermore, two IL-12-oHSVs with equivalent transcriptional activity differed in IL-12 protein production in vivo, and the differences in IL-12 protein levels were reflected in immune infiltrate activity changes as well as tumor growth suppression differences between the IL-12-oHSVs. C002-treated tumors exhibited sustained IL-12 production with improved dendritic cells, monocyte-macrophage activity (MHCII, CD80/CD86 upregulation) and a polyfunctional Th1-cell response in the tumor infiltrates. Conclusion: These results suggest that transgene protein production differences between oHSVs in vivo, in addition to replication differences, can impact OV-therapeutic activity.

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

The quest for effective immunotherapies against malignant peripheral nerve sheath tumors: Is there hope?

Immune-based therapies represent a new paradigm in the treatment of multiple cancers, where they have helped achieve durable and safe clinical responses in a growing subset of patients. While a wealth of information is available concerning the use of these agents in treating the more common malignancies, little has been reported about the use of immunotherapies against malignant peripheral nerve sheath tumors (MPNSTs), a rare form of soft tissue sarcoma that arises from the myelin sheaths that protect peripheral nerves. Surgical resection has been the mainstay of therapy in MPNSTs, but the recurrence rate is as high as 65%, and chemotherapy is generally ineffective. The immune contexture of MPNSTs, replete with macrophages and a varying degree of T cell infiltration, presents multiple opportunities to design meaningful therapeutic interventions. While preliminary results with macrophage-targeting strategies and oncolytic viruses are promising, identifying the subset of patients that respond to immune-based strategies will be a milestone. As part of our effort to help advance the use of immunotherapy for MPNSTs, here we describe recent insights regarding the immune contexture of MPNSTs, discuss emerging immune-based strategies, and provide a brief overview of potential biomarkers of response.

Oncolytic virus-driven immune remodeling revealed in mouse medulloblastomas at single cell resolution.

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.

Genetically modified IL2 bone-marrow-derived myeloid cells reprogram the glioma immunosuppressive tumor microenvironment.

Gliomas are one of the leading causes of cancer-related death in the adolescent and young adult (AYA) population. Two-thirds of AYA glioma patients are affected by low-grade gliomas (LGGs), but there are no specific treatments. Malignant progression is supported by the immunosuppressive stromal component of the tumor microenvironment (TME) exacerbated by M2 macrophages and a paucity of cytotoxic T cells. A single intravenous dose of engineered bone-marrow-derived myeloid cells that release interleukin-2 (GEMys-IL2) was used to treat mice with LGGs. Our results demonstrate that GEMys-IL2 crossed the blood-brain barrier, infiltrated the TME, and reprogrammed the immune cell composition and transcriptome. Moreover, GEMys-IL2 extended survival in an LGG immunocompetent mouse model. Here, we report the efficacy of an in vivo approach that demonstrates the potential for a cell-mediated innate immunotherapy designed to enhance the recruitment of activated effector T and natural killer cells within the glioma TME.

Opportunities and challenges of combining adoptive cellular therapy with oncolytic virotherapy.

The use of oncolytic viruses (OVs) and adoptive cell therapies (ACT) have independently emerged as promising approaches for cancer immunotherapy. More recently, the combination of such agents to obtain a synergistic anticancer effect has gained attention, particularly in solid tumors, where immune-suppressive barriers of the microenvironment remain a challenge for desirable therapeutic efficacy. While adoptive cell monotherapies may be restricted by an immunologically cold or suppressive tumor microenvironment (TME), OVs can serve to prime the TME by eliciting a wave of cancer-specific immunogenic cell death and inducing enhanced antitumor immunity. While OV/ACT synergy is an attractive approach, immune-suppressive barriers remain, and methods should be considered to optimize approaches for such combination therapy. In this review, we summarize current approaches that aim to overcome these barriers to enable optimal synergistic antitumor effects.

Myeloid cell heterogeneity in the tumor microenvironment and therapeutic implications for childhood central nervous system (CNS) tumors.

Central nervous system (CNS) tumors are the most common type of solid tumors in children and the leading cause of cancer deaths in ages 0-14. Recent advances in the field of tumor biology and immunology have underscored the disparate nature of these distinct CNS tumor types. In this review, we briefly introduce pediatric CNS tumors and discuss various components of the TME, with a particular focus on myeloid cells. Although most studies regarding myeloid cells have been done on adult CNS tumors and animal models, we discuss the role of myeloid cell heterogeneity in pediatric CNS tumors and describe how these cells may contribute to tumorigenesis and treatment response. In addition, we present studies within the last 5 years that highlight human CNS tumors, the utility of various murine CNS tumor models, and the latest multi-dimensional tools that can be leveraged to investigate myeloid cell infiltration in young adults and children diagnosed with select CNS tumors.

Pediatric versus adult high grade glioma: Immunotherapeutic and genomic considerations.

High grade gliomas are identified as malignant central nervous tumors that spread rapidly and have a universally poor prognosis. Historically high grade gliomas in the pediatric population have been treated similarly to adult high grade gliomas. For the first time, the most recent classification of central nervous system tumors by World Health Organization has divided adult from pediatric type diffuse high grade gliomas, underscoring the biologic differences between these tumors in different age groups. The objective of our review is to compare high grade gliomas in the adult versus pediatric patient populations, highlighting similarities and differences in epidemiology, etiology, pathogenesis and therapeutic approaches. High grade gliomas in adults versus children have varying clinical presentations, molecular biology background, and response to chemotherapy, as well as unique molecular targets. However, increasing evidence show that they both respond to recently developed immunotherapies. This review summarizes the distinctions and commonalities between the two in disease pathogenesis and response to therapeutic interventions with a focus on immunotherapy.

Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics.

T cells redirected to cancer cells either via a chimeric antigen receptor (CAR-T) or a bispecific molecule have been breakthrough technologies; however, CAR-T cells require individualized manufacturing and bispecifics generally require continuous infusions. We created an off-the-shelf, single-dose solution for achieving prolonged systemic serum levels of protein immunotherapeutics via adeno-associated virus (AAV) gene transfer. We demonstrate proof of principle in a CD19+ lymphoma xenograft model using a single intravenous dose of AAV expressing a secreted version of blinatumomab, which could serve as a universal alternative for CD19 CAR-T cell therapy. In addition, we created an inducible version using an exon skipping strategy and achieved repeated, on-demand expression up to at least 36 weeks after AAV injection. Our system could be considered for short-term and/or repeated expression of other transgenes of interest for noncancer applications.

Cardiorespiratory management of Duchenne muscular dystrophy: emerging therapies, neuromuscular genetics, and new clinical challenges.

The life-limiting complications of Duchenne muscular dystrophy (DMD) include loss of lung function and progressive cardiomyopathy; when patients are treated with assisted ventilation, cardiac function becomes the main determinant of survival. Therapy for DMD is changing rapidly, with the emergence of new genetic and molecular therapeutic options, the proliferation of which has fostered the perception that DMD is a potentially curable disease. However, data for respiratory and cardiac outcomes are scarce and available evidence is not uniformly positive. Patients who share a dystrophin (DMD) genotype can have highly divergent cardiorespiratory phenotypes; genetic modifiers of DMD gene expression are a probable cause of respiratory and cardiac phenotypic variability and discordance. In this Personal View, we provide an overview of new and emerging DMD therapies, highlighting the limitations of current research and considering strategies to incorporate cardiorespiratory assessments into clinical trials. We explore how genetic modifiers could be used to predict cardiorespiratory natural history and how manipulation of such modifiers might represent a promising therapeutic strategy. Finally, we examine the changing role of respiratory physicians, cardiologists, and intensive care clinicians on the frontline of a challenging new clinical landscape.

Clinical outcomes and efficacy of stereotactic body radiation therapy in children, adolescents, and young adults with metastatic solid tumors.

OBJECTIVE: The objective of this study is to report disease outcomes and toxicity with the use of stereotactic body radiation therapy (SBRT) in the treatment of pediatric metastatic disease. METHODS: All pediatric and adolescent young adult (AYA) patients' who received SBRT were included between the years 2000 and 2020. Study endpoints included local control (LC), progression-free survival (PFS), overall survival (OS), cumulative incidence (CI) of death or local failure and toxicity. The end points with respect to survival and LC were calculated using the Kaplan-Meier estimate. The cumulative incidence of local failure was calculated using death as a competing risk. RESULTS: 16 patients with 36 lesions irradiated met inclusion criteria and formed the study cohort. The median OS and PFS for the entire cohort were 17 months and 15.7 months, respectively. The 1 year OS for the entire cohort was 75%. The 6- and 12 month local control was 85 and 78%, respectively. There were no local failures in irradiated lesions for patients who received a BED10≥100 Gy. Patients who were treated with SBRT who had ≤5 metastatic lesions at first recurrence had a superior 1 year OS of 100 vs 50% for those with >5 lesions. One patient (6.3%) experienced a Grade 3 central nervous system toxicity. CONCLUSION: LC was excellent with SBRT delivered to metastatic disease, particularly for lesions receiving a BED10≥100 Gy. High-grade toxicity was rare in our patient population. Patients with five or fewer metastatic sites have a significantly better OS compared to >5 sites. ADVANCES IN KNOWLEDGE: This study demonstrates that SBRT is safe and efficacious in the treatment of pediatric oligometastatic disease.

Immunotherapies for Pediatric Solid Tumors: A Targeted Update.

There are encouraging signs in our collective progress to leverage the immune system to treat pediatric cancers. Here, we summarize interim successes in cancer immunotherapy and opportunities to translate from the adult world to pediatrics, and highlight challenges that could benefit from additional development, focusing on solid tumors. Just a decade ago, other than antibodies targeting disialoganglioside (GD2) in neuroblastoma, pediatric cancer immunotherapy was mostly relegated to obscure preclinical studies in a few academic labs. Today there are numerous clinical trials of a variety of antibody, cellular, gene, and viral therapies and vaccines designed to either promote antitumor immunity or specifically attack validated immunotherapy targets. Understanding those targets and their pediatric relevance is paramount. While much work is underway to evaluate the utility of numerous immunologic targets, the lack of regulatory approvals is emblematic of the challenges that remain. Herein we focus our review on the most promising targeted immunotherapies in clinical trials for children.