<div>AbstractPurpose:<p>Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape.</p>Experimental Design:<p>We studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT.</p>Results:<p>Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells.</p>Conclusions:<p>These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.</p></div>
<p>Supplementary Table 1:Tumors maintained expression of subgroup genes previously identified as unique to corresponding human MB subtypes, SHH and Group 3 tumors, respectively. Supplementary Table 2: Flow cytometric antibody panel.</p>
Abstract INTRODUCTION One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in non-glioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with anti-tumor activity. METHODS We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 weeks, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD FACSCanto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism. RESULTS Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (< 1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, p < 0.0001). CONCLUSION Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.
Abstract INTRODUCTION Trogocytosis is the exchange of membrane components from one cell to another. During T cell activation, dendritic cell (DC) and T cell membranes become anchored around the TCR/MHC complex and their costimulatory molecules. Part of the DC membrane is pulled away, taken in, and educates the T cell to recognize foreign antigens. We have utilized this phenomenon to isolate and expand a select population of T cells that have undergone this interaction. HYPOTHESIS: (1) By selecting for cells that have undergone trogocytosis, we are enriching for a tumor-specific population of T cells and can improve anti-tumor responses in the setting of adoptive cellular transfer (ACT). (2) This approach represents a reliable method to detect tumor-specific antigens across numerous types of brain cancers. METHODS Murine DCs are pulsed with KR158B glioma RNA and then stained with a proliferation dye such as Cell Trace Violet. These cells are co-cultured overnight with CD3+ splenocytes of vaccinated mice and IL-2. The cells are then sorted into T cells that have undergone trogocytosis (CD3+ CTV+) and those that have not (CD3+CTV-) and placed back into co-culture with fresh dendritic cells for expansion of tumor-specific T cells and/or restimulation assay. Additionally, T cells are spectratyped and phenotyped by flow cytometry. RESULTS Cells that had undergone trogocytosis (CD3+CTV+) demonstrated superior IFNγ secretion and enriched for CD4+ and CD8+ central memory and effector subsets compared to cells that had not undergone trogocytosis (CD3+CTV-). Spectratyping analysis revealed CD3+CTV+ cells enriched for Vβ 5.1/5.2 and Vβ 6, two families we have determined to be crucial for anti-tumor efficacy. CONCLUSIONS This method of selection and enrichment of tumor-specific T cells has promising implications for the enhancement of ACT. This method can be used to elucidate tumor-specific antigens that are unique to an individual’s cancer fingerprint.
Abstract Mutations in tumor neoantigens have shown promise in immunotherapies for many cancers, yet pediatric brain tumors, which typically have a lower mutational burden, offer fewer opportunities to capitalize on these targeted therapies. Studies indicate that medulloblastoma (MB) and brain stem gliomas (BSG) originate from abnormal reactivation of post-natal developmental processes. This led us to investigate whether proteins expressed during the early post-natal development of the mouse cerebellum and brainstem could be powerful antigens to fight MB and BSG, respectively. We performed both in vitro and in vivo studies in mice with the RNA of developmental antigens from the brain stem and cerebellum in an adoptive cellular therapy (ACT) platform. We evaluated the reactivity and therapeutic efficacy of these treatments as sustainable antigenic targets against BSG and MBs. We demonstrate that T cells activated towards these non-mutated, tissue-specific developmental antigens can recognize distinct subtypes of MB and BSG, and activate specifically without cross-reacting to the normal brain. This conferred a survival benefit in established orthotopic models of these pediatric brain tumor types. Our research exhibits the value developmental antigens can offer when serving as tumor rejection antigens in MB and BSG.
Abstract A Liquid Like Solid (LLS) 3D culture system in a convenient microtiter plate format enables long-duration culture of patient derived microtumors (>30 days), in situ confocal imaging, 3D cytotoxic drug studies, inclusion of T cells, and measurements of T cell migration, infiltration, and killing. Introduction: Cancer is a disease in 3-dimensions and there is a desperate need for new tools and infrastructure to study immuno-oncology treatment methods in 3D. Fabrication of microtumors using 3D printing in LLS media comprised of soft granular microgels (3-5 μm crosslinked polyacrylamide (PAA) microgel particles: 7.5% PAA) facilitate precise arrangement of detailed assemblies of extra-cellular matrix components and cells (1), including: epithelial cells (breast ATCC MCF10A), stem cells (bone marrow MSC), cancer cells (breast ATCC MCF7, prostate ATCC-PC3, osteosarcoma ATCC-MG63, melanoma ATCC-A375, primary glioblastoma, and osteosarcoma), fibroblasts (ThermoFisher dermal fibroblasts C0045C), endothelial cells (ThermoFisher HUVECs C0035C), and CD4+/CD8+ T cells (PBMC and Jurkat E6-1). Methods: Long term culture was enabled through the design, development, and validation of a modular perfusion system that uses passive negative pressure within a 96-well microtiter plate format to transport liquid growth medium, drugs (doxorubicin and puromycin), antibodies (aPD1 J43 clone, aCD3, aCD28), growth factors, FBS, and metabolic waste without disturbing the spatial organization and positioning of the experiments (i.e. 3D orientation is preserved and the packed bed of microgel particles remain solid). The perfusion velocities are precisely controlled to between 1-100 nm/s by setting the negative pressure, and complete exchange of liquid media can be tailored from hours to days. Results: Real-time imaging in these 3D assays is performed using in situ confocal microscopy under controlled perfusion. Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved over the first 72 hours. Immunohistochemistry with Ki-67 and PCNA staining indicated active cell proliferation of the tumoroids after 28 days of continuous culture. Tracking of activated CD8+ T cells revealed super-diffusive motion in the presence of 3D tumors within a range of 250 µm. Activated T cell migration speeds have been measured to be between 1.3 and 2.0 μm/s in the 3D LLS media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Conclusions: This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ 3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors. References: 1. Battacharjee et al. Science Advances Sept. 2015 1:8 Citation Format: Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Juan M. Urueña, Duane A. Mitchell, Catherine T. Flores, Steven C. Ghivizzani, C Parker Gibbs, Padriac P. Levings, Colin J. Anderson, W Gregory Sawyer. 3D in vitro system for immuno-oncology: Real-time imaging of drug delivery, tumoroids, and immune cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 35.
Dendritic cell (DC) plays crucial role in eliciting anti-tumor immune response through both innate and adaptive immune system. Our group has developed an adoptive cellular therapy (ACT) against high grade gliomas, which significantly improves survival in murine models of CNS malignancies. Our studies have shown that the efficacy is associated with the observed increase in intratumoral dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC).1 However, tumors develop diverse mechanisms to restrict DC function to evade immune surveillance. In this study, we will address the various DC dysfunctional mechanism in both primary and ACT escaped tumors.
Methods
ACT treatment was administered to mice bearing the KR158B glioblastoma cell line to get ACT escaped tumor. The mice received 9 Gy of irradiation prior to the transfer of hematopoietic stem cells and tumor-reactive T cells, followed by three doses of BMDC vaccine. Tumor associated DCs sorted from primary and ACT escaped tumors were subjected to assess DC function or gene expression profiling. T cell proliferation, T cell activation markers, and IFNγ secretion were measured by flow cytometry and ELISA as a readout for DC-T cell co-culture functional assays. Brain tumor slices were prepared using a vibratome, and the conditioned medium from the slice culture was used to pre-treat DCs to determine the impact of brain tumor secretion on DCs.
Results
Functional evidence demonstrated that DCs from ACT-escaped tumors is impaired in activating tumor-reactive T cells. By comparing the gene expressing profiles between tumor associated DCs in primary and ACT escaped tumors,we found a significant decrease in cDC marker genes, antigen assembly genes, and MHC molecules in ACT-escaped tumor DCs, compared to primary tumor DCs, suggesting a decreased cDC population and impaired antigen presentation in ACT-escaped tumor DCs. To determine whether secreted factors from tumor microenvironment that drives DC dysfunction, conditioned medium(CM) from tumor brain slices were tested in inducing DC dysfunction in vitro. CM from primary tumor brain slices suppressed DC function compared to healthy brain slice CM. Further results showed that non-tumor cell-derived secretion from the slices drives DC dysfunction in primary tumors. Strikingly, unlike primary tumor brain CM, ACT-escaped tumor brain slice CM did not decrease DC function capacity, suggesting different tumor-driven DC dysfunctional mechanisms in primary and ACT-escaped tumors.
Conclusions
The primary glioma tumor drives DC dysfunction through non-tumor cell-derived secretion, whereas the ACT-escaped tumor employs a different mechanism involving cDC exclusion and impaired MHC expression and antigen loading.
References
Wildes TJ, et al. Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma. Clin Cancer Res, 2018; 24(16): 3955–3966.
Ethics Approval
The study is approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and are covered under protocol number IACUC202100000053. All participants were given informed consent before taking part.
The purpose of this study is to investigate the effects of exercise on cancer progression, metastasis, and underlying mechanisms in an orthotopic model of murine prostate cancer. C57BL/6 male mice (6–8 wk of age) were orthotopically injected with transgenic adenocarcinoma of mouse prostate C-1 cells (5 × 10 5 ) and randomly assigned to exercise ( n = 28) or a non-intervention control ( n = 31) groups. The exercise group was given voluntary access to a wheel 24 h/day for the duration of the study. Four mice per group were serially killed on days 14, 31, and 36; the remaining 38 mice (exercise, n = 18; control, n = 20) were killed on day 53. Before death, MRI was performed to assess tumor blood perfusion. Primary tumor growth rate was comparable between groups, but expression of prometastatic genes was significantly modulated in exercising animals with a shift toward reduced metastasis. Exercise was associated with increased activity of protein kinases within the MEK/MAPK and PI3K/mTOR signaling cascades with subsequent increased intratumoral protein levels of HIF-1α and VEGF. This was associated with improved tumor vascularization. Multiplex ELISAs revealed distinct reductions in plasma concentrations of several angiogenic cytokines in the exercise group, which was associated with increased expression of angiogenic and metabolic genes in the skeletal muscle. Exercise-induced stabilization of HIF-1α and subsequent upregulation of VEGF was associated with “productive” tumor vascularization with a shift toward suppressed metastasis in an orthotopic model of prostate cancer.
BACKGROUND: Pediatric brain tumors are the number one cause of solid cancer death in children, and thus necessitate the development of novel targeted therapeutics. To enhance the feasibility of cancer immunotherapy, off-the-shelf vaccination strategies have been proposed, but have proven to be only weakly immunogenic. Consequently we have designed a novel, translatable nanoparticle (NP) vaccine that can be embedded with immunomodulatory modifications to generate robust anti-tumor responses against murine models for glioblastoma (GBM) and medulloblastoma (MB). OBJECTIVES: We sought to assess if RNA-NP vaccines would induce superior immune responses against GBM and MB by stimulating intracellular pathogen recognition receptors (PRRs) while simultaneously downregulating regulatory pathways. RESULTS: We screened commercially available and clinically translatable NP formulations and determined that the cationic liposome DOTAP was the most superior NP for delivery of RNA to antigen presenting cells (APCs) in vitro and in vivo. Afterwards, we verified that these particles preserve RNA stability over time, and induce in vivo gene expression. These RNA-NPs elicited potent T cell immunity, superior to peptide vaccines formulated in Complete Freund's Adjuvant (CFA) and induced anti-tumor efficacy in adoptive cellular therapy platforms in murine GBM and MB models. We showed dramatic upregulation of activation markers including CD80 and CD86 and demonstrated abrogation of regulatory markers such as programmed death-ligand1 (PD-L1) when RNA-NPs were administered in combination with anti-PD-L1 monoclonal antibodies. Finally, we demonstrated enhanced immune responses through incorporation of immunomodulatory RNAs encoding for pathogen associated molecular patterns (PAMPs) derived from hepatitis C. CONCLUSION: Since clinically translatable RNA-NP vaccines can deliver combinatorial therapies using a single delivery platform, they represent a novel platform for inducing potent nontoxic immunity against intracranial tumors that can be harnessed to provide a more effective and specific therapy critical in improving clinical outcomes for children affected by these malignancies.
