A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling.

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.

Implementing Continuous Quality Improvement in an Integrated Community Urology Practice: Lessons Learned.

OBJECTIVE: To determine the effectiveness of 2 different continuous quality improvement interventions in an integrated community urology practice. We specifically assessed the impact of audited physician feedback on improving physicians' adoption of active surveillance for low-risk prostate cancer (CaP) and adherence to a prostate biopsy time-out intervention. MATERIALS AND METHODS: The electronic medical records of Genesis Healthcare Partners were analyzed between August 24, 2011 and September 30, 2020 to evaluate the performance of 2 quality interventions: audited physician feedback to improve active surveillance adoption in low-risk CaP patients, and audited physician feedback to promote adherence to an electronic medical records embedded prostate biopsy time-out template. Physician and Genesis Healthcare Partners group adherence to each quality initiative was compared before and after each intervention type using ANOVA testing. RESULTS: For active surveillance, we consistently saw an increase in active surveillance adoption for low risk CaP patients in association with continuous audited feedback (P < .001). Adherence to the prostate biopsy time-out template improved when audited feedback was provided (P < .001). CONCLUSION: The implementation of clinical guidelines into routine clinical practice remains challenging and poses an obstacle to the improvement of United States healthcare quality. Continuous quality improvement should be a dynamic process, and in our experience, audited feedback coupled with education is most effective.

Leveraging the Electronic Medical Record Improves Prostate Cancer Clinical Staging in a Community Urology Practice.

INTRODUCTION: Clinical tumor staging is an important component of risk stratification, which is central in assessment and treatment of patients with prostate cancer. We evaluated the potential of an examination based tumor staging template embedded within the electronic medical record to improve consistency and clarity of clinical tumor staging. METHODS: We conducted a retrospective analysis before template implementation (January to March 2017), followed by prospective analysis after implementation (April to August 2017). Physicians were educated on use and importance of the staging template prior to implementation. Assessment of digital rectal examination clarity included confident-explicit (cT stage documented), confident-implicit (stage inexplicit, cT stage interpreted) and unconfident (inability to discern cT stage). Clarity and consistency of tumor staging before and after template implementation were compared using a chi-square test. RESULTS: A total of 573 biopsies were analyzed: 234 before template use (40%) and 339 after (60%). In men at risk for prostate cancer explicit staging increased from 16% to 60% (p <0.001) following implementation of the staging template. Overall staging (explicit plus implicit) increased (from 74% to 92%, p <0.001), while unconfident staging decreased (from 26% to 8%, p <0.001) after implementation. In men with positive biopsies (309) explicit staging increased (from 29% to 64%, p <0.001) and overall staging increased (from 76% to 92%, p <0.001), while unconfident staging decreased (from 24% to 8%, p <0.001). CONCLUSIONS: Accurate prostate cancer clinical staging is important in risk stratification and treatment. We demonstrate the ability of a standardized, electronic medical record embedded, American Joint Committee on Cancer based template to improve the consistency and clarity of clinical staging in prostate cancer.

Three-year Active Surveillance Outcomes in a Contemporary Community Urology Cohort in the United States.

OBJECTIVES: To determine the 3-year outcomes of men with prostate cancer managed with active surveillance (AS) in a cohort of geographically diverse community-based urology practices. AS is the management of choice for a majority of men with lower risk prostate cancer.1,2,3 Little is known about the contemporary "real-world" follow-up and adherence rates in the most common setting of urologic care, community (private) practice.4 METHODS: We retrospectively evaluated outcomes for men diagnosed between January 1, 2013 and May 31, 2014 with National Comprehensive Cancer Network (NCCN) very low, low and intermediate risk prostate cancer who selected AS in 9 large community urology practices. We used univariate and multivariate analyses to describe associations between race, age, insurance status, family history, comorbidity, clinical stage, Gleason score, NCCN risk-group, and PSA density with discontinuation of AS. RESULTS: Five hundred and forty-eight men on AS were followed for a median of 3.35 years. 89% (492) continued to follow-up with diagnosing practice. 32% (171) discontinued AS. On multivariate analysis, increasing NCCN risk classification (Hazard ratio [HR] 1.65, P = 0.02 and HR 2.09, P < 0.01 for low and intermediate risk vs very low risk) was significantly associated with discontinuation. Among those who discontinued AS, surgery and radiation were utilized equally (47% and 53%, respectively, P = 0.48). CONCLUSION: In this community-based cohort of men on AS, a minority was lost to follow-up and adherence to AS was similar to other reports. Disease characteristics more than sociodemographic characteristics correlated with adherence to AS, while surgery and radiotherapy were utilized equally among those discontinuing AS, both suggesting guideline concordant practice of medicine.

Change in prostate cancer presentation coinciding with USPSTF screening recommendations at a community-based urology practice.

OBJECTIVE: The benefits of prostate-specific antigen (PSA)-based prostate cancer screening are controversial. We sought to determine the change in prostate cancer presentation coinciding with the release of the United States Preventative Services Task Force recommendations against screening in a high-volume community-based urology practice. METHODS: Characteristics of men presenting for an elevated PSA at a community urology practice from August 2011 to August 2015 were queried from a prospectively collected database. A retrospective analysis of presenting PSA, Gleason grade at biopsy, and prostatectomy as well as clinical and pathologic stage was performed. Kruskal-Wallis rank sum and chi-square tests were used for analysis. RESULTS: Referrals for elevated PSA decreased from 933 in year 1 to 816 by year 4 (12.5% decrease) with a concomitant reduction in biopsies performed in newly referred men from 461 to 356 (22.8% decrease, P = 0.02). The proportion of men presenting with PSAs>10 increased from 28.1% to 36.8% (P = 0.009). First-time biopsy-positivity rate increased from 48.4% to 62.4% with a rise in the proportion having Gleason≥7 from 51.6% to 69.7% (P = 0.0001). Of the 578 men who underwent radical prostatectomy, there was a 19.4% increase in Gleason≥7 tumors (P = 0.01). CONCLUSIONS: Our findings demonstrate a decrease in elevated PSA referrals, increase in PSA at the time of referral, decrease in detection of low-risk disease, and increase in detection of intermediate-/high-risk disease in a high-volume, multisite, community-based urology practice, coinciding with the United States Preventative Services Task Force recommendations against PSA screening.

Aminoacyl tRNA Synthetase--Interacting Multifunctional Protein 1 Activates NK Cells via Macrophages In Vitro and In Vivo.

Aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1) has been reported to have antitumor effects in various tumor models. However, mechanisms by which AIMP1 ameliorates tumorigenesis are not well understood. As NK cells are a major cell type involved in antitumor activities and AIMP1 is known to activate professional APCs, we determined whether AIMP1 induced NK cell activation directly or via these APCs. AIMP1 induced the expression of surface activation markers on murine NK cells in total splenocytes, although AIMP1 did not directly induce these activation markers of NK cells. The inductive effect of AIMP1 on NK cell activation disappeared in macrophage-depleted splenocytes, indicating that macrophages were required for the AIMP1-induced activation of NK cells. Furthermore, coculture experiments showed that AIMP1 activated NK cells in the presence of isolated macrophages, but failed to activate NK cells when cultured alone or with dendritic cells or B cells. Although AIMP1 significantly promoted TNF-α production by macrophages, the secreted TNF-α partially affected the NK cell activation. Transwell cocultivation analysis revealed that direct contact between macrophages and NK cells was required for the AIMP1-induced NK cell activation. In addition, AIMP1 significantly enhanced cytotoxicity of NK cells against Yac-1 cells. Furthermore, the in vivo administration of AIMP1 also induced NK cell activation systemically with a macrophage-dependent manner. Importantly, AIMP1 dramatically reduced the lung metastasis of melanoma cells, which was mediated by NK cells. Taken together, our results show that AIMP1 induces antitumor responses by NK cell activation mainly via macrophages.

Prostate Biopsy Complications: A Dual Analysis.

OBJECTIVE: To measure prostate needle biopsy (PNB)-associated complications and place of treatment: inpatient hospitalization and outpatient treatment. An electronic medical record (EMR) data query is compared to a patient questionnaire survey. MATERIALS AND METHODS: A total of 2410 patients underwent 2588 biopsies and were evaluated for PNB-associated complications. Two approaches were used: (1) EMR analysis based on International Classification of Diseases, Ninth Revision, and Current Procedural Terminology coding and chart review; and (2) patient-reported questionnaire and chart review validation. Serious complications were defined as any complication leading to a related hospitalization, visit to the emergency department (ED), urgent care (UC), or doctor's office within 30 days of the biopsy. RESULTS: The EMR study revealed 69 (2.67%) serious complications leading to either hospitalization or treatment at an ED, UC, or doctor's office. Thirty serious complications led to hospitalization (1.16%), 14 patients (0.54%) were treated at the ED, 1 was managed at a UC (0.04%), and 24 (0.93%) were treated at the doctor's office. Of the 847 (35.1%) questionnaires considered appropriate for analysis, 36 (4.25%) reported treatment in either the hospital, ED, UC, or doctor's office. Nine patients (1.06%) reported being hospitalized within 30 days of the procedure, whereas 27 patients (3.19%) were treated in an outpatient setting, 8 (0.94%) at the ED, 3 (0.35%) at a UC, and 16 (1.89%) at the doctor's office. CONCLUSION: Our dual analysis study indicates a slightly greater than 1% incidence of hospitalization due to serious complications following PNB. Serious complications appear to be more frequently managed outside the hospital setting (ED, UC, and doctor's office).

Active Surveillance of Prostate Cancer in a Community Practice: How to Measure, Manage, and Improve?

OBJECTIVE: To measure past active surveillance (AS) adoption rates, institute the best practice, and measure the AS adoption rates following implementation. We report our findings over a 3-year period. METHODS: Patient prostate needle biopsy and treatment data from the period August 2011 to August 2014 were retrieved from an integrated electronic medical records (Allscripts) and stored in a Microsoft Access database for analysis. Structured data were queried using the automated software program WizMD and unstructured data were abstracted by manual review. AS adoption was calculated according to four different selection criteria. Between 2013 and 2014, physicians at Genesis Healthcare Partners (GHP) underwent an educational training program on the University of California, San Diego/GHP AS best practice for managing low-risk prostate cancer patients and were provided report cards on their AS adoption and comparative reporting. RESULTS: AS adoption increased for the 3 years of the study. AS adoption for all newly diagnosed patients managed at GHP increased from 12.9% to 14.74%. AS adoption for patients with low-risk prostate cancer (as defined by the National Comprehensive Cancer Network) increased from 31.90% to 58.46% from year 1 to year 3 of the study (P < .001), and AS adoption for the most strict (restrictive) criteria increased from 43.75% to 82.61% (P < .001) after the educational and comparative reporting intervention. CONCLUSION: These data highlight the potential benefit of physician education and comparative reporting to enhance AS adoption. AS adoption rates vary according to selection criteria used for analysis. Carefully selected outcomes from evidence-based guidelines have the potential to enhance medical quality.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions.

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

Method for co-cluster analysis in multichannel single-molecule localisation data.

We demonstrate a combined univariate and bivariate Getis and Franklin's local point pattern analysis method to investigate the co-clustering of membrane proteins in two-dimensional single-molecule localisation data. This method assesses the degree of clustering of each molecule relative to its own species and relative to a second species. Using simulated data, we show that this approach can quantify the degree of cluster overlap in multichannel point patterns. The method is validated using photo-activated localisation microscopy and direct stochastic optical reconstruction microscopy data of the proteins Lck and CD45 at the T cell immunological synapse. Analysing co-clustering in this manner is generalizable to higher numbers of fluorescent species and to three-dimensional or live cell data sets.

Immunity to Trop-1, a newly identified breast cancer antigen, inhibits the growth of breast cancer in mice.

This study describes the immunotherapeutic properties of vaccines that encode tumor-associated calcium signal transducer-1 (Trop-1), a newly identified breast cancer antigen, in mice with breast cancer. Previously we found that Trop-1 was over-expressed in cellular breast cancer vaccines that were highly enriched for cells that induced therapeutic CTL-mediated immune responses in mice with breast cancer, as compared with non-enriched vaccines. In this study, to determine if the expression of Trop-1 by cells in the enriched vaccine was responsible for its therapeutic benefits, an expression plasmid that specified the Trop-1 gene was transfected into the LM fibroblast cells, which was then used as a vaccine. To augment their immunogenic properties, the fibroblasts were genetically modified before Trop-1 DNA-transfer to secrete IL-2 and to express allogeneic MHC class I H-2K(b)-determinants. Mice with established breast cancer treated solely by immunization with fibroblasts modified to express Trop-1 developed CD8(+) cell-mediated immunity to the breast cancer cells. The immunity was sufficient to prolong the survival of mice with established breast cancer. In some instances, the immunity was sufficient to result in rejection of the tumor; the mice remained tumor free more than 60 days.

A human monoclonal antibody against insulin-like growth factor-II blocks the growth of human hepatocellular carcinoma cell lines in vitro and in vivo.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.

Crystallization and preliminary X-ray analysis of the complexes between a Fab and two forms of human insulin-like growth factor II.

Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab-IGF-II complexes (M64-F02-IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 A. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 107, c = 111.5 A, and also diffracted X-rays to 2.2 A resolution.

Enhancing cellular cancer vaccines.

Various strategies have been used to generate cellular cancer vaccines with the expectation that they will become an effective part of the overall management of cancer patients. However, with few notable exceptions, immunization has not resulted in significant long-term therapeutic benefits. Tumor growth has continued and patient survival has been at best only modestly prolonged. One possible explanation is that as only a small proportion of the constituents of malignant cells are "tumor specific" and the vast majority are the products of nonantigenic, normal "housekeeping" genes, the immune response in patients immunized with cellular cancer vaccines is not sufficient to result in tumor rejection. Here, we review and characterize various types of cellular cancer vaccines. In addition, in a mouse breast cancer model system, we describe a unique strategy designed to enrich cellular vaccines for cells that induce tumor immunity. Numerous advantages and disadvantages of cancer immunotherapy with cellular vaccines are also presented.

Gene transfer of AIMP1 and B7.1 into epitope-loaded, fibroblasts induces tumor-specific CTL immunity, and prolongs the survival period of tumor-bearing mice.

T helper type 1 (Th1) cell-mediated immune responses play various roles in cellular immunity, including inducing cytotoxic T lymphocytes (CTLs) and they have been shown to be crucial in cancer immunotherapy. Previously, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) stimulated antigen-presenting cells to secrete IL-12, leading to enhanced Th1 cell responses. In this study, as a way of enhancing antigen-specific Th1 responses, mouse fibroblasts (H-2(b)) were genetically modified to express an AIMP1 and a costimulatory B7.1 (Fb/AIMP1/B7.1). Fb/AIMP1/B7.1 cells were then loaded with an ovalbumin epitope as a model antigen (Fb/AIMP1/B7.1/OVA), and tested to determine if they induced OVA-specific CTLs in C57BL/6 mice (H-2(b)). Immunization with Fb/AIMP1/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EG7 tumor cells, but not against other H-2(b) tumor cells. The levels of the cytotoxic response in the immunized mice with Fb/AIMP1/B7.1/OVA cells were significantly higher than the responses in mice immunized with other cell constructs. CD8(+) T cells were a major cell-type of OVA-specific antitumor immunity induced by Fb/AIMP1/B7.1/OVA cells. Furthermore, treatment with Fb/AIMP1/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. These results indicate that AIMP1-secreting, epitope-loaded fibroblasts efficiently induce antigen-specific CTL responses in mice.

Immunity to growth factor receptor-bound protein 10, a signal transduction molecule, inhibits the growth of breast cancer in mice.

This study describes the application of a unique strategy to identify breast cancer antigens [tumor-associated antigen (TAA)]. In a mouse model, the strategy led to the identification of growth factor receptor-bound protein 10 (Grb10) as a newly identified TAA. Grb10 is a signal transduction molecule associated with multiple transmembrane tyrosine kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice with nonenriched vaccines. The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line C3H/He origin (H-2(k)), into LM mouse fibroblasts (H-2(k)). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell population, however, incorporated cDNA fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was used, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were overrepresented in the enriched vaccines. One, the gene for Grb10, was approximately 100-fold overrepresented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor rejection.

Enhanced immunity to intracerebral breast cancer in mice immunized with a cDNA-based vaccine enriched for immunotherapeutic cells.

Structural differences between malignant and nonmalignant cells of the same individual form the basis of clinical immunotherapeutic strategies. Previously, we reported the therapeutic properties of a vaccine prepared by transfer of a cDNA-expression library from breast cancer cells into a highly immunogenic allogeneic fibroblast cell line where genes specifying an array of breast cancer antigens were expressed. Here, we report the application of this cell-based vaccination strategy for breast cancer metastatic to the brain. As relatively few cells in the vaccine were expected to have incorporated cDNA fragments specifying breast cancer antigens (most specify normal cellular constituents), an enrichment strategy was employed to increase the proportion of immunotherapeutic cells. Enhanced immunity to the neoplasm mediated predominantly by CD4+, CD8+, and NK/LAK cells was generated in the spleens of mice injected intracerebrally into the tumor bed with cells from the enriched vaccine, which translated into prolonged survival. Regulatory T cells (CD4+CD25+Foxp3+-positive) were relatively deficient in the spleen cells from tumor-bearing mice injected intracerebrally with the enriched vaccine.

Prolonged survival of mice with established intracerebral glioma receiving combined treatment with peroxisome proliferator-activated receptor-gamma thiazolidinedione agonists and interleukin-2-secreting syngeneic/allogeneic fibroblasts.

OBJECT: In this study the authors explored the benefits of treating C57B1/6 mice with an established intracerebral glioma by combining immunotherapy with interleukin (IL)-2-secreting syngeneic/allogeneic fibroblasts administered into the tumor bed along with the chemotherapeutic agent pioglitazone, a thiazolidinedione (TZD). The TZDs are agonists of the peroxisome proliferator-activated receptor-gamma. They have been found to exert antiproliferative effects on several transformed cell lines. Data from prior studies by these authors have revealed the immunotherapeutic properties of the IL-2-secreting fibroblasts in treating intracerebral gliomas in mice. METHODS: The sensitivity of GL261 glioma cells and primary astrocytes to pioglitazone was determined in vitro by incubating the cells with increasing amounts of the drug. Viability was assessed by measuring lactate dehydrogenase release, and effects on metabolism were determined by measuring superoxide production and levels of superoxide dismutase. The GL261 cells were injected intracerebrally into C57B1/6 mice, followed by treatment with pioglitazone either orally or intracerebrally into the tumor bed. The effect of the combined therapy was determined by injecting C57B1/6 mice with an established intracerebral GL261 glioma with IL-2-secreting allogeneic fibroblasts and pioglitazone directly into the tumor bed through a unique cannula system. Pioglitazone was found to induce cell death in GL261 glioma cells grown in vitro while causing only modest damage to astrocytes. The application of pioglitazone also resulted in a significantly greater induction of cellular superoxide in glioma cells than in astrocytes, which can activate apoptotic pathways. Pioglitazone administered intracerebrally (p < 0.05) but not orally was found to prolong survival in mice harboring an intracerebral glioma. Synergistic effects of combination therapy on prolonging survival were found in mice receiving both pioglitazone and IL-2-secreting fibroblasts (p < 0.005, compared with untreated animals). Pioglitazone induces metabolic and oxidative stresses that are tolerated by astrocytes but not glioma cells, which could account for selective vulnerability and increased sensitivity to IL-2, suggesting potential for the use of this Food and Drug Administration-approved drug in the treatment of brain tumors. CONCLUSIONS: The data indicate the beneficial effects of combination therapy using pioglitazone and immunotherapy in mice harboring intracerebral glioma.