Expression of Intracellular Reactive Oxygen Species in Hematopoietic Stem Cells Correlates with Time to Neutrophil and Platelet Engraftment in Patients Undergoing Autologous Bone Marrow Transplantation.

Reactive oxygen species (ROS) play important roles in hematopoiesis and regulate the self-renewal, migration, and myeloid differentiation of hematopoietic stem cells (HSCs). This study was conducted to determine whether ROS levels in donor HSCs correlate with neutrophil and platelet engraftment in patients after bone marrow transplantation. Cryopreserved HSC samples from 51 patients who underwent autologous transplantation were studied. Levels of intracellular ROS were assessed by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in the CD45+/CD34+ HSC population. Colony forming unit assays were performed on HSCs isolated from the ROShigh and ROSlow populations to assess the differentiation potential of these 2 cell subsets. Distinct populations of ROShigh and ROSlow cells were evident in all patient samples. The median percentage of ROShigh expressing HSCs in the study cohort was 75.8% (range, 2% to 95.2%). A significant correlation was identified between the percentage of ROShigh stem cells present in the hematopoietic progenitor cells collected by apheresis product infused and the time to neutrophil engraftment (P < .001, r = -.54), as well as time to plt20, plt50, and plt100 (P < 0.001; r = -.55, -.59, and -.56 respectively). The dose of CD34+/ROShigh/kg infused also inversely correlated with a shorter time to neutrophil engraftment; time to engraftment for patients receiving > or ≤3 × 106 cells/kg was 11.5 days (range, 9 to 23) versus 14 days (range, 10 to 28), respectively (P = .02). The dose of ROShigh HSCs delivered did not correlate with platelet engraftment. Collectively, these data suggest that the dose of ROShigh stem cells delivered to patients may predict time to neutrophil engraftment after autologous transplantation.

Measurement of global DNA methylation levels by flow cytometry in mouse fibroblasts.

DNA Methylation, 5meC, is an epigenetic modification that acts as an important regulator of genomic stability and gene expressivity. Genome-wide changes in methylation have been associated with lineage-specific changes in gene expression profiles during development and in some cell-based pathologies, including oncogenesis. Cost-effective and rapid platforms for the detection of changes in the global levels of methylation are of value for the investigation of the processes that regulate methylation. Flow cytometry allows rapid and quantitative analysis of epitopes within a large number of cells. We have recently optimised the conditions required for valid detection of 5meC by immunofluorescence microscopy. These studies showed that immunological detection of 5meC requires the sequential denaturation of chromatin by a brief period of acidification followed by a partial tryptic digestion step. We have assessed the reliability of flow cytometry for the detection of changes in 5meC when coupled with this optimised epitope retrieval strategy. This study provides support for the use of high throughput screening of 5meC by flow cytometry for the analysis of the epigenetic regulation of important cell transitions.

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 µl whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.

Induction of antitumor immunity using autologous tumor proteins is an attractive approach to cancer therapy. However, better methods and stimulants to present these autologous proteins back to the immune system are needed. Here, we identify streptavidin as a novel carrier protein and stimulant, and test the efficacy of both syngeneic (rat) and autologous vaccines (dogs) using streptavidin in combination with reduced soluble tumor proteins. Initial syngeneic vaccine studies in the 9L rat glioma model were used to optimize vaccine dose and selectivity. Cytokine and blood analysis was used to monitor the response. Rats receiving two vaccinations of syngeneic tumor vaccine demonstrated a statistically significant (P < 0.05) survival advantage compared with controls (adjuvant only). Notably, vaccination also led to remission rates of between 30% and 60% in the aggressive 9L glioma model. Antibodies to streptavidin were detected in the serum of vaccinated rats; however, antibody levels did not correlate with the response. The cytokine TNF-α was upregulated in vaccine-treated rats, whereas ICAM1 was downregulated. After engraftment, vaccinated rats maintained CD4(+), CD8(+) T cells, and total lymphocyte levels closer to normal baseline than those in the controls. Twenty-five dogs treated with autologous vaccine preparations using streptavidin as a stimulant showed no adverse reactions, irrespective of additional chemotherapy and other medications. In this study, we developed a novel method for producing syngeneic and autologous vaccines using streptavidin selectivity and immunogenicity. These vaccines show efficacy in the 9L glioma rat model. Safety was also demonstrated in canine patients presenting with cancer treated with autologous vaccine.

Cotargeting of epidermal growth factor receptor and PI3K overcomes PI3K-Akt oncogenic dependence in pancreatic ductal adenocarcinoma.

PURPOSE: PI3K-Akt is overexpressed in 50% to 70% of pancreatic ductal adenocarcinoma (PDAC). The hypothesis of this study is that PI3K and EGFR coinhibition may be effective in PDAC with upregulated PI3K-Akt signaling. EXPERIMENTAL DESIGN: Multiple inhibitors were tested on five PDAC cell lines. EGFR inhibitor (EGFRi)-resistant cell lines were found to have significantly overexpressed AKT2 gene, total Akt, and pAkt. In vitro erlotinib-resistant (ER) cell models (BxPC-ER and PANC-ER) with highly constitutively active PI3K-Akt were developed. These and their respective parent cell lines were tested for sensitivity to erlotinib, IGFIR inhibitor NVP-AEW541 (AEW), and PI3K-alpha inhibitor NVP-BYL719 (BYL), alone or in combination, by RTK-phosphoarray, Western blotting, immunofluorescence, qRT-PCR, cell proliferation, cell cycle, clonogenic, apoptosis, and migration assays. Erlotinib plus BYL was tested in vivo. RESULTS: Erlotinib acted synergistically with BYL in BxPC-ER (synergy index, SI = 1.71) and PANC-ER (SI = 1.44). Treatment of ER cell lines showing upregulated PI3K-Akt with erlotinib plus BYL caused significant G1 cell-cycle arrest (71%, P < 0.001; 58%, P = 0.003), inhibition of colony formation (69% and 72%, both P < 0.001), and necrosis and apoptosis (75% and 53%, both P < 0.001), more so compared with parent cell lines. In primary patient-derived tumor subrenal capsule (n = 90) and subcutaneous (n = 22) xenografts, erlotinib plus BYL significantly reduced tumor volume (P = 0.005). Strong pEGFR and pAkt immunostaining (2+/3+) was correlated with high and low responses, respectively, to both erlotinib and erlotinib plus BYL. CONCLUSION: PDAC with increased expression of the PI3K-Akt pathway was susceptible to PI3K-EGFR coinhibition, suggesting oncogenic dependence. Erlotinib plus BYL should be considered for a clinical study in PDAC; further evaluation of pEGFR and pAkt expression as potential positive and negative predictive biomarkers is warranted.