Temozolomide is the most widely used chemotherapy for patients with glioblastoma (GBM) despite the fact that approximately half of treated patients have temozolomide resistance and all patients eventually fail therapy. Due to the limited efficacy of existing therapies, immunotherapy is being widely investigated for patients with GBM. However, initial immunotherapy trials in GBM patients have had disappointing results as monotherapy. Therefore, combinatorial treatment strategies are being investigated. Temozolomide has several effects on the immune system that are dependent on mode of delivery and the dosing strategy, which may have unpredicted effects on immunotherapy. Here we summarize the immune modulating role of temozolomide alone and in combination with immunotherapies such as dendritic cell vaccines, T-cell therapy, and immune checkpoint inhibitors for patients with GBM.
How tissue-specific anatomical distribution and phenotypic specialization are linked to protective efficacy of memory T cells against reinfection is unclear. Here, we show that lung environmental cues program recently recruited central-like memory cells with migratory potentials for their tissue-specific functions during lethal respiratory virus infection. After entering the lung, some central-like cells retain their original CD27hiCXCR3hi phenotype, enabling them to localize near the infected bronchiolar epithelium and airway lumen to function as the first line of defense against pathogen encounter. Others, in response to local cytokine triggers, undergo a secondary program of differentiation that leads to the loss of CXCR3, migration arrest, and clustering within peribronchoarterial areas and in interalveolar septa. Here, the immune system adapts its response to prevent systemic viral dissemination and mortality. These results reveal the striking and unexpected spatial organization of central- versus effector-like memory cells within the lung and how cooperation between these two subsets contributes to host defense.
Immune cell migration is critical for cellular immunotherapy efficacy in the treatment of glioblastoma (GBM). Sarcosine is a non-toxic metabolite that is associated with a migratory phenotype in prostate cancer cells. We utilized sarcosine to increase migration of dendritic cells (DC) and T cells to increase efficacy of cellular immunotherapy. HYPOTHESIS: Sarcosine increases cellular migration and will improve tumor outcomes when combined with cellular immunotherapy platforms in the treatment of GBM. DC and T cell migration was evaluated in vitro using transwell plates and in vivo using flowcytometry and immunofluorescence microscopy. The impact of sarcosine loaded cells on tumor outcomes was measured by testing a DC vaccine platform in the treatment of murine tumor models. Genomic expression of cytokines on sarcosine treated cells was tested using RT-PCR. Cells were efficiently loaded with sarcosine by simply adding sarcosine to the culture media (20mM). DCs and T cells loaded with sarcosine demonstrated increased migration in vitro (p<0.0001). Mice treated with DC vaccination in the setting of sarcosine demonstrated significantly increased DC migration to draining lymph nodes and spleens (p<0.05). Gene expression analysis demonstrated that sarcosine caused upregulation of cytokines (IFNg,Xcl1,Fasl,Csf2,CCL19,Bmp2,IFNa2 and IL27) and downregulation of other cytokines (Ccl22,IL1b,Cxcl3,Ccl5,IL9 and IL18)(p<0.05). Vaccination with sarcosine loaded OVA-DCs led to increased proliferation of antigen specific T cells compared to regular OVA-DC vaccination (p<0.05). B16F10-OVA tumor bearing animals treated with sarcosine loaded OVA-DC vaccines had significantly reduced tumor growth and prolonged survival compared to animals treated with OVA-DC vaccines without sarcosine (p<0.0001). Sarcosine loaded DC vaccines resulted in suppressed tumor growth and improved survival in a murine model. These effects are partially mediated by changes in cytokine expression in sarcosine loaded DCs. Further experiments testing sarcosine loaded T cells and the mechanism of increased migration in sarcosine loaded immune cells are underway.
Abstract The role of immunology is now critical for the understanding of cancer pathophysiology and treatment. Dendritic cells (DCs) are immune cells that serve the unique purpose of bridging innate immunity with the adaptive immune response. Owing to their specialised role, they have been leveraged to deliver immunotherapy for cancer. Key Concepts Dendritic cells are antigen presenting cells that present antigens to naïve T cells serving as a link between the innate and adaptive immune response. Dendritic cells can be generated from peripheral blood mononuclear cells, loaded with an antigen and given back to the patient as a vaccine. Dendritic cells can various antigens including non-infectious antigens related to disease such as cancer. Several dendritic cell vaccines have been tested for efficacy in cancer and one is currently FDA approved for prostate cancer. Dendritic cell vaccines have been tested in patients with cancer with promising results.
OBJECTIVE The objective of this study was to develop a murine system for the delivery of laser interstitial thermotherapy (LITT) with probe-based thermometry as a model for human glioblastoma treatment to investigate thermal diffusion in heterogeneous brain tissue. METHODS First, the tissue heating properties were characterized using a diode-pumped solid-state near-infrared laser in a homogeneous tissue model. The laser was adapted for use with a repurposed stereotactic surgery frame utilizing a micro laser probe and Hamilton syringe. The authors designed and manufactured a stereotactic frame attachment to work as a temperature probe stabilizer. Application of this novel design was used as a precise method for real-time thermometry at known distances from the thermal ablative center mass during murine LITT studies. RESULTS Temperature measurements were achieved during LITT that verified the direct thermometry capability of the system without the need for MR-based thermal monitoring. Application of multiple stereotactic design iterations led to an accurately reproducible surgical laser ablation procedure. Histological staining confirmed precise thermal ablation and controllable lesion size based on time and temperature control. Treatment of a syngeneic intracranial glioma model highly resistant to conventional therapy resulted in a modest survival benefit. CONCLUSIONS The authors have successfully developed a murine model system of LITT with direct in situ thermometry for investigation into the effects of thermal ablation and combinatorial treatments in murine brain tumor models.
Abstract Dendritic cell (DC) vaccine efficacy is directly related to the efficiency of DC migration to the lymph node after delivery to the patient. In this research we discovered increasing cell migration by utilizing sarcosine improved anti-tumor efficacy. We hypothesized that sarcosine induced cell migration was due to chemokine or cytokine signaling. METHODS To generate DC vaccines, DCs were harvested from bone marrow of wild type C57BL/6 mice and electroporated with OVA-mRNA. Human DCs were isolated from PBMCs. DCs were treated with sarcosine at 20mM. OT-I T cells were isolated from transgenic mice and injected intravenously into B16F10-OVA tumor bearing mice. Following T cell transfer, DC vaccines were injected intradermal. In vitro migration was analyzed via transwell migration assay. In vivo migration was evaluated by flow-cytometry and immunofluorescence microscopy. Gene expression in RNA was investigated in DCs via RT-PCR and Nanostring. RESULTS Sarcosine significantly increases human and murine DC migration in vitro. In vivo murine model, sarcosine-loaded DCs had significantly increased migration to both the lymph nodes and spleen after intradermal delivery. B16F10-OVA tumor bearing mice were treated with the sarcosine-loaded DC vaccines resulted in a significant survival advantage over control and naïve DC vaccines. Gene expression in RNA was investigated in DCs. CXCR2,CXCL3 and CXCL1 were found to be upregulated in sarcosine-loaded DCs. Further metabolic analysis demonstrated the upregulation of cyclooxygenase-1 and Pik3cg. In vitro DC migration in presence of CXCR2 neutralizing antibody showed sarcosine induced migration was abrogated by adding the CXCR2 neutralizing antibody in both human and murine DCs. Animals that treated with sarcosine-loaded DC showed significantly better tumor control compares to the animals receiving anti-CXCR2 antibody one hour before DC injection. CONCLUSION Sarcosine increases the migration of murine and human DCs via the CXC chemokine pathway. This platform can be utilized to improve existing DC vaccine strategies.
Precision health provides an unprecedented opportunity to improve the assessment of infant nutrition and health outcomes. Breastfeeding is positively associated with infant health outcomes, yet only 58.3% of children born in 2017 were still breastfeeding at 6 months. There is an urgent need to examine the application of precision health tools that support the development of public health interventions focused on improving breastfeeding outcomes.
Immunotherapy with adoptive transfer T cells is an effective treatment for cancer. We have recently demonstrated the ability to overcome chemotherapy resistance in a mouse model of malignant glioma with an adoptive T cell. This platform is dependent on effective migration of cells to the tumor site. Therefore, tracking of transferred cells is critical to the evaluation of the immunotherapy efficacy. The objective of this project was to establish a novel method to track immune cells in vivo using 13C-labeling of nucleic acids. Human T cells were isolated from peripheral blood mononuclear cells (PBMCs). The T cells were stimulated and were grown in glucose-free media supplemented with 13C-labeled glucose in different concentrations (5.5mM, 10mM, 15mM) for 5-7 days. DNA was extracted at various time points and cellular dilutions. Samples were digested and Tandem mass spectrometry (MS/MS) scan events were performed for the 4 nucleosides as well as their most abundant 13C labeled isotope or a plus 5 mass shift resulting from 13C5 incorporations into the ribose backbone. An extracted Ion Chromatogram (EIC) was done for the MS/MS the mass fragments and the peak areas were analyzed. 13C-labeling was identified in all samples including the most diluted and the latest time point. The 13C detection was confirmed to be from labeled T cells as naturally abundant 13C does not incorporate into all of the carbons of the DNA ribose backbone and would not result in a 5 mass shift. Adenosine 13C labeling was the most sensitive. dA/idA ratio in different dilutions (1, 0.25, 0.1, 0.01, 1x10-2, 1x10-3, 1x10-4) were 0.26, 2.47, 2.51, 13.33, 85.74, 118, 190.01. Even cells removed from 13C labeled glucose for 7 days demonstrated detectable levels of 13C-labeled DNA. 13C-labeled glucose can be used to label immune cell DNA for sensitive in vivo tracking.
