Probing Spatial Myeloid Heterogeneity in Glioblastoma

https://openworks.mdanderson.org/sumexp22/1015/thumbnail.jp

Preclinical Efficacy and Safety Profile of Allometrically Scaled Doses of Doxycycline Used to Turn “On” Therapeutic Transgene Expression from High-Capacity Adenoviral Vectors in a Glioma Model

Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity ?gutless? adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible ?Tet-On? activation system that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we evaluated the safety and efficacy of Food and Drug Administration (FDA)?approved doses of the tetracycline doxycycline (DOX) allometrically scaled for rats. DOX initiates the expression of Flt3L, which has been shown to recruit dendritic cells to the brain tumor microenvironment?an integral first step in the development of antitumor immunity. The data revealed a highly safe profile surrounding these human-equivalent doses of DOX under an identical therapeutic window as proposed in the clinical trial. This was confirmed through a neuropathological analysis, liver and kidney histopathology, detection of neutralizing antibodies, and systemic toxicities in the blood. Interestingly, we observed a significant survival advantage in rats with GBM receiving the 300?mg/day equivalent dosage of DOX versus the 200?mg/day equivalent. Additionally, rats rejected ?recurrent? brain tumor threats implanted 90 days after their primary brain tumors. We also show that DOX detection within the plasma can be an indicator of optimal dosing of DOX to attain therapeutic levels. This work has significant clinical relevance for an ongoing phase I clinical trial in humans with primary GBM and for other therapeutic approaches using Tet-On transactivation system in humans.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140103/1/hgtb.2015.168.pd

Utilizing Tissue Microarrays for Medium-Throughput Validation of Antibodies Against Immune Markers

https://openworks.mdanderson.org/sumexp21/1201/thumbnail.jp

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration

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Intestinal Toxicity to Ctla-4 Blockade Driven by Il-6 and Myeloid Infiltration

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs

Modeling of CNS Cancer with a Focus on the Immune Component

The knowledge surrounding cancers of the central nervous system remains poorly developed, in particular with regard to the immune component. The works contained in this thesis look at craniopharyngioma, glioblastoma, and several forms of brain metastasis. While some attention is given to the tumor cells themselves, as well as the patient setting which these studies model, the immune component of disease progression and treatment plays a strong role in each and is the primary focus of the works contained. Craniopharyngioma is a relatively rare tumor in adults. Although histologically benign, it can be locally aggressive and may require additional therapeutic modalities to surgical resection. In the first set of experiments contained within, multiplatform analyses including next generation sequencing, chromogenic and in situ hybridization, immunohistochemistry, and gene amplification were used to profile craniopharyngiomas (n=6) to identify frequent therapeutic targets. Sixty-seven percent of patients had the BRAF V600E missense mutation, frequent in the papillary craniopharyngioma subtype. One patient had a missense mutation in the WNT pathway, specifically a mutation in CTNNB1 associated with the adamantinomatous subtype. Craniopharyngiomas lacked microsatellite instability, had relatively low tumor mutational burden, but did express PD-L1 protein, indicating potential use therapeutic use for immune checkpoint inhibition. We identified mutations not previously described, including an E318K missense mutation in the MITF gene, an R1407 frameshift in the SETD2 gene of the PIK3CA pathway, R462H in the NF2 gene, and a I463V mutation in TSC2. Two patients testing positive for epidermal growth factor receptor (EGFR) expression were negative for the EGFRvIII variant. Herein, we identified several alterations such as those in BRAF V600E and PD-L1, which may be considered as targets for combination therapy of residual craniopharygiomas. We hope that these insights may lead to better treatment of patients with this disease in the future. Novel therapeutic strategies, including immunotherapeutics, targeting glioblastoma (GBM) often fail in the clinic, at least partly because available preclinical models do not recapitulate the human disease. To address this challenge in our second set of experiments, we took advantage of our previously developed spontaneous Qk/trp53/Pten (QPP) triple-knockout model of human GBM and compared its immune microenvironment components with those of patient-derived tumors in effort to determine whether this model might provide an opportunity for gaining insights into tumor physiopathology as well as for preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice as well as from GBM patients revealed intratumoral immune components that were predominantly myeloid cells (e.g., monocytes, macrophages, and microglia) with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found that there were more neutrophils, T and NK cells in the implanted model. Neutrophils, T and NK cells were increased in abundance in samples derived from human high-grade glioma (HGG) compared to those derived from low grade glioma (LGG). Overall, our data demonstrate that our implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid dominant nature of the tumor microenvironment of human gliomas. Our model provides a suitable tool for investigating the complex immune compartment of gliomas and it may contribute to a better understanding of the resistance of human glioblastoma to currently available immunotherapeutics. Given that we established the QPP model as viable for immune studies we next sought to pursue a therapeutic target in the form of Arginase-1 using this system. An average GBM has roughly 30% infiltration of myeloid cells, the highest reported case at 70% infiltration, with some variation dependent on subtype. When we consider this fact in combination with the advent and success of immunotherapies in similar cancer types, the logical next step follows that myeloid cells, which are of the immune lineage, have the potential to clear this aberrant growth. Known for their incredible phagocytic capacity, myeloid cells are among the first responders to an injury to control pathogens and clear apoptotic cells from the site of an insult. Myeloid cells, through poorly understood mechanisms, undergo a switch after 3-5 days from attacking pathogens and clearing dead cells to secretion of growth cytokines to promote wound healing. We posit these myeloid cells may be tricked into erroneously supporting the tumor cells in an attempt to “heal the wound” instead of mounting an immune response. The enzyme responsible for this switch from pathogen response to wound healing is called Arginase-1 (Arg1). When Arg1 is upregulated, it has a two-pronged effect, the first is creation of peroxynitrite which is actively immunosuppressive to both myeloid and T-cells. The second is an increase in ornithine when Arg1 cleaves arginine into ornithine and urea, which can be used to create the building blocks for cellular growth as well as to create collagen, which is necessary for wound repair and found in high levels in brain tumors. Our hypothesis is that through manipulation of the Arg1 axis in myeloid cells we will increase anti-tumor response. We show the groundwork for evaluating whether or not this is a viable therapeutic target in GBM as well as assess currently available compounds for inhibition or Arg1 in the CNS. It is a truism in cancer medicine that 90% of deaths are caused by cancer metastasis. While unfortunately difficult to collect information on, there are datasets that suggest the trend in increased metastatic cancer deaths has outpaced primary cancer deaths in recent decades. Metastasis to the brain is a large cause of mortality and roughly 300,000 cases are diagnosed annually. In an effort to address this urgent patient need we aimed to build a library of metastatic tumor models that were representative of the patient population and determine whether the models could be used for preclinical utility. We first assessed the growth patterns, latency and penetrance of our models and found that in general models tended to grow or not with little impact on survival from starting cell number. We came across several modeling problems including lack of engraftment and extracranial growth of tumors that presented challenges for modeling certain types of metastases to the CNS. We next chose to put several of our models through a battery of therapeutic regimens including Csfr1 inhibition, checkpoint blockade in the forms of anti-Pd-1 and anti-Lag-3 monoclonal antibodies, as well as radiotherapy. We used these single agent studies to inform upon potentially synergistic combinations that would then inform upon clinical trials

Assessing the role of STAT3 in DC differentiation and autologous DC immunotherapy in mouse models of GBM.

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors

CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study

Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy