A Therapeutic Vaccine Targeting Rat BORIS (CTCFL) for the Treatment of Rat Breast Cancer Tumors.

Cancer testis antigens are ideal for tumor immunotherapy due to their testis-restricted expression. We previously showed that an immunotherapeutic vaccine targeting the germ cell-specific transcription factor BORIS (CTCFL) was highly effective in treating aggressive breast cancer in the 4T1 mouse model. Here, we further tested the therapeutic efficacy of BORIS in a rat 13762 breast cancer model. We generated a recombinant VEE-VRP (Venezuelan Equine Encephalitis-derived replicon particle) vector-expressing modified rat BORIS lacking a DNA-binding domain (VRP-mBORIS). Rats were inoculated with the 13762 cells, immunized with VRP-mBORIS 48 h later, and then, subsequently, boosted at 10-day intervals. The Kaplan-Meier method was used for survival analysis. Cured rats were re-challenged with the same 13762 cells. We demonstrated that BORIS was expressed in a small population of the 13762 cells, called cancer stem cells. Treatment of rats with VRP-BORIS suppressed tumor growth leading to its complete disappearance in up to 50% of the rats and significantly improved their survival. This improvement was associated with the induction of BORIS-specific cellular immune responses measured by T-helper cell proliferation and INFγ secretion. The re-challenging of cured rats with the same 13762 cells indicated that the immune response prevented tumor growth. Thus, a therapeutic vaccine against rat BORIS showed high efficacy in treating the rat 13762 carcinoma. These data suggest that targeting BORIS can lead to the elimination of mammary tumors and cure animals even though BORIS expression is detected only in cancer stem cells.

Adoptive T-Cell Therapy in Advanced Colorectal Cancer: A Systematic Review.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US. For the vast majority of patients with advanced CRC (ie, for those in whom metastatic tumors are unresectable), treatment is palliative and typically involves chemotherapy, biologic therapy, and/or immune checkpoint inhibition. In recent years, the use of adoptive T-cell therapy (ACT), leveraging the body's own immune system to recognize and target cancer, has become increasingly popular. Unfortunately, while ACT has been successful in the treatment of hematological malignancies, it is less efficacious in advanced CRC due in part to a lack of productive immune infiltrate. This systematic review was conducted to summarize the current data for the efficacy and safety of ACT in advanced CRC. We report that ACT is well tolerated in patients with advanced CRC. Favorable survival estimates among patients with advanced CRC receiving ACT demonstrate promise for this novel treatment paradigm. However, additional stage I/II clinical trials are needed to establish the efficacy and safety of ACT in patients with CRC.

Immunological and psychosocial functioning in parents of children with cancer.

PURPOSE: Research has shown that parents of children with cancer exhibit an altered immune profile compared to parents of healthy children, reflective of increased susceptibility to illness. These parents are also at risk for poorer psychosocial outcomes and quality of life. The current study compares peripheral blood cell analyses and psychosocial self-reports from parents of children being treated for cancer (n = 21) to parents of healthy children (n = 30). METHODS: A blood sample was drawn from parents to analyze immune profiles. Parents also completed the Perceived Stress Scale (PSS), Medical Outcomes Study Short Form-36 (MOS), and Patient-Reported Outcomes Measurement Information System Short Form v1.0 Emotional Distress-Anxiety 8a, and Emotional Distress-Depression 8a (PROMIS). Mann-Whitney U tests and independent samples t-tests were conducted to examine differences in outcomes between parent groups. RESULTS: Parents of children with cancer exhibited higher monocyte percentages in their peripheral blood compared to peers with healthy children. Parents of children with cancer also reported poorer psychosocial outcomes: higher perceived stress, higher anxiety and depression symptoms, more role disability resulting from emotional problems, poorer general and mental health, and poorer social functioning. CONCLUSION: These findings support research that has shown a direct effect of chronic stress on the immune system. Symptoms reported by parents of children with cancer indicate unmet psychosocial needs that could potentially affect long-term health. Given the central role of parents in their children's cancer care, it is compelling to address and work to improve parent immunological and psychosocial well-being.

Longitudinal Changes in Sleep: Associations with Shifts in Circulating Cytokines and Emotional Distress in a Cancer Survivor Population.

BACKGROUND: Sleep disturbances are associated with numerous mood disorders. Similarly, anxiety and depression are associated with modulation of the psychoneuroimmune (PNI) axis. This study hypothesized that changes in both monitored and self-reported measures of sleep would relate to changes in circulating cytokine levels in an emotionally distressed population of cervical cancer survivors. METHODS: Biospecimens, patient-reported outcome (PRO) measures, and actigraphy were collected from cervical cancer survivors enrolled in a biobehavioral clinical trial. Longitudinal changes over a 4-month period were examined. Sleep time measured by actigraphy and PRO were analyzed for correlative changes with emotional distress and serum cytokines (n = 71). RESULTS: Longitudinal change in the actigraph measure of sleep time was inversely associated with changes in depression and anxiety (test for linear trend, p = 0.02 and p = 0.05 respectively), as well as acute-phase response/pro-inflammatory cytokines (test for linear trend, p = 0.003, interleukin (IL)-2; 0.022, IL-1ß; 0.0002, IL-6; and 0.049, tumor necrosis factor α). Conversely, changes in self-reported sleep problems were related to an increase in depression and anxiety (p = 0.001 and p = 0.01 respectively), the T helper 2 (Th2) cytokine IL-5 (p = 0.027), and the counter-regulatory cytokine IL-10 (0.016). CONCLUSION: This study showed that an increase in sleep time or decrease in sleep problems corresponded with a reduction in self-reported emotional distress and attenuation of pro-inflammatory, Th2, and counter-regulatory cytokines. Our results support sleep measurement as a meaningful biobehavioral variable in cancer survivorship. This study also indicates that sleep investigators should be aware that choice of methodology might influence concordance with different classes of immune parameters.

Cytokine profiling: variation in immune modulation with preterm birth vs. uncomplicated term birth identifies pivotal signals in pathogenesis of preterm birth.

OBJECTIVES: To assess deviations in longitudinally measured cytokines with preterm birth (PTB). METHODS: Prospective longitudinal study targeting 80 subjects. Phlebotomy specimens for broad panel of cytokine analysis were obtained at three time (T) intervals: first trimester (T1: 8-14 weeks' gestation), second trimester (T2: 18-22 weeks' gestation), and third trimester (T3: 28-32 weeks' gestation). Important demographics and outcomes were tracked. Data were stratified and the target groups were analyzed as follows: "Uncomplicated" (delivered ≥37 weeks) or "Preterm Birth" (<37 weeks). Generalized Linear Modeling determined rate of change T1-T3 by outcome. RESULTS: Complete data replete with phlebotomy at all three visits were obtained on 80 women. Birth outcomes were as follows: 11 Uncomplicated Term Birth (UTB), 28 PTB, 4 low birth weight (LBW), 16 OB complications (OBC), 11 current infections (IFN), and 10 mixed complications (MC=2 or more of the above). 28 PTB were compared to 11 uncomplicated term deliveries. In both groups, T helper type 1 (TH1) cytokine (IL-1ß), pleiotrophic pro-inflammatory cytokine (IL-6), and counter-regulatory cytokine (IL-10) responses decreased over gestation, but rates of change in IL-1ß, IL-6, and IL-10 were significantly different. Stratification of women by smoking status additionally demonstrated significant variance in immune status over the course of pregnancy. CONCLUSIONS: Women delivering PTB demonstrated significant differences in cytokine trajectory over pregnancy; these data further validate key role played by immune regulation in directing pregnancy outcome. Likewise, smoking impacts longitudinal trajectory of cytokines over pregnancy.

Anticancer Mechanisms in Two Murine Bone Marrow-Derived Dendritic Cell Subsets Activated with TLR4 Agonists.

Dendritic cells (DCs) are well-known for their functions in orchestrating the innate and adaptive arms of immune defense. However, under certain conditions, DCs can exert tumoricidal activity. We have elucidated the mechanism of tumor suppression by TLR4-activated bone marrow-derived DCs (BMDCs) isolated from BALB/c mice. We identified that two distinct subsets of BMDCs (CD11b+CD11c+I-A/Eint and CD11b+CD11c+I-A/Ehigh) have different cytotoxic mechanisms of action. The cytotoxicity of the former subset is mediated through NO and reactive oxygen species and type I IFN (IFN-ß), whereas the latter subset acts only through IFN-ß. TLR4 agonists, LPS or pharmaceutical-grade ImmunoMax, activate CD11c+ BMDCs, which, in turn, directly kill 4T1 mouse breast cancer cells or inhibit their proliferation in an MHC-independent manner. These data define two populations of BMDCs with different mechanisms of direct cytotoxicity, as well as suggest that the I-A/Eint subset could be less susceptible to counteracting mechanisms in the tumor microenvironment and support investigation of similar subsets in human DCs.

Standard preoperative use of nonopioid multi-modal medications for patients undergoing mastectomy with immediate reconstruction and the effect on postoperative opioid needs.

Standardized nonopioid preoperative protocol effects perioperative opioids. Combined use of acetaminophen, pregabalin, celecoxib, and transdermal scopolamine (APCS), in mastectomy with immediate subpectoral reconstruction procedures. Retrospective (2014-2017) cohort study (n = 305) examined treatment groups; APCS, no treatment (NONE), and partial combination APCS (OTHER), employing multivariable gamma regression models controlling preoperative and perioperative variables, examining postoperative opioid use (oral morphine equivalents, OME) and hospital stay (hours, LOS). APCS group had a 25% statistical reduction in OME total vs OTHER, a 12% statistical reduction in LOS vs OTHER, and 11% statistical reduction in LOS vs NONE. Standardized nonopioid preoperative protocol provides insight into perioperative opioid use.

Metabolite Responsive Nanoparticle-Protein Complex.

Stimuli-responsive polymers are an efficient means of targeted therapy. Compared to conventional agents, they increase bioavailability and efficacy. In particular, polymer hydrogel nanoparticles (NPs) can be designed to respond when exposed to a specific environmental stimulus such as pH or temperature. However, targeting a specific metabolite as the trigger for stimuli response could further elevate selectivity and create a new class of bioresponsive materials. In this work we describe an N-isopropylacrylamide (NIPAm) NP that responds to a specific metabolite, characteristic of a hypoxic environment found in cancerous tumors. NIPAm NPs were synthesized by copolymerization with an oxamate derivative, a known inhibitor of lactate dehydrogenase (LDH). The oxamate-functionalized NPs (OxNP) efficiently sequestered LDH to produce an OxNP-protein complex. When exposed to elevated concentrations of lactic acid, a substrate of LDH and a metabolite characteristic of hypoxic tumor microenvironments, OxNP-LDH complexes swelled (65%). The OxNP-LDH complexes were not responsive to structurally related small molecules. This work demonstrates a proof of concept for tuning NP responsiveness by conjugation with a key protein to target a specific metabolite of disease.

Relationship between social support, quality of life, and Th2 cytokines in a biobehavioral cancer survivorship trial.

OBJECTIVE: Benefits of social support (SS) during cancer survivorship are complex. This study examines change in SS over time in cervical cancer (CXCA) survivors who have completed definitive treatment and how changing SS impacts quality of life (QOL) and T-helper type 2 (Th2) cytokines. METHODS: We conducted a randomized trial in 204 CXCA survivors to test if psychosocial telephone counseling (PTC) could improve QOL compared to usual care (UC). Although PTC did not target SS, data were collected at baseline, 4 and 9 months post-enrollment using the Medical Outcomes Survey Social Support scale. Biospecimens were collected to investigate associations with patient-reported outcomes. Data were analyzed using multivariate linear models and stepwise regression. RESULTS: Participants' mean age was 43. PTC participants experienced increasing SS compared to UC at 4 months (PTC-UC = 5.1; p = 0.055) and 9 months (PTC-UC = 6.0; p = 0.046). Higher baseline SS and increasing SS were independently associated with improved QOL at 4 and 9 months after adjusting for patient characteristics (p < 0.05). Differences between study arms were not statistically significant. Improvements in QOL at 4 months were observed with increases in emotional/informational and tangible SS. Increasing SS predicted significant longitudinal decreases in IL-4 and IL-13 at 4 months that were larger in the PTC arm (interactions p = 0.041 and p = 0.057, respectively). CONCLUSION: Improved SS was significantly associated with improved QOL independent of patient characteristics and study arm. Decreasing Th2 cytokines with increasing SS and QOL are consistent with a biobehavioral paradigm in which modulation of the chronic stress response is associated with shifts in immune stance.

Method for Dynamically Detecting Secretions from Single Cells Using a Nanopore.

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.

Validation of PROMIS emotional distress short form scales for cervical cancer.

OBJECTIVES: Cervical cancer patients are at high risk for emotional distress. In this study we evaluate the PROMIS emotional distress-Depression and -Anxiety Short Forms for assessing depression and anxiety in a cervical cancer population. METHODS: A 15-item questionnaire was used in a cervical cancer biobehavioral randomized clinical trial, testing psychosocial telephone counseling (PTC) against usual care (UC). It was administered to 204 patients prior to randomization, four months post-enrollment, and nine months post-enrollment, together with legacy measures of depression. The short forms were evaluated in patients participating in this study over three time points for internal consistency, convergent validity, and responsiveness to change over time. RESULTS: Overall, 45% and 47% of patients scored in the moderate to severe range for anxiety and depression, respectively. Internal consistency coefficients were ≥ 0.95 at baseline, 4 months, and 9 months for depression and anxiety. The average inter-item correlation was 0.65 and 0.73 at baseline assessment for depression and anxiety, respectively. The depression short form T-score was correlated with legacy distress scales ranging from 0.44-0.76, and the anxiety short form ranging from 0.45-0.78. The depression short form demonstrated sensitivity to change as patients randomized to the counseling intervention reported greater improvement over time in depression (p = 0.014), and a nonsignificant improvement in anxiety, compared to the patients receiving usual care. CONCLUSIONS: The PROMIS depression and anxiety short forms reliably and validly assess cervical cancer-specific emotional distress, capture salient features of distress in this population, and perform as well or better than legacy measures.

Surgical Outcomes for Mastectomy Patients Receiving Neoadjuvant Chemotherapy: A Propensity-Matched Analysis.

OBJECTIVE: To evaluate the risk of neoadjuvant chemotherapy for surgical morbidity after mastectomy with or without reconstruction using 1:1 matching. BACKGROUND: Postoperative surgical complications remain a potentially preventable event for breast cancer patients undergoing mastectomy. Neoadjuvant chemotherapy is among variables identified as contributory to risk, but it has not been rigorously evaluated as a principal causal influence. METHODS: Data from American College of Surgeons National Surgical Quality Improvement Program (2006-2012) were used to identify females with invasive breast cancer undergoing planned mastectomy. Surgical cases categorized as clean and undergoing no secondary procedures unrelated to mastectomy were included. A 1:1 matched propensity analysis was performed using neoadjuvant chemotherapy within 30 days of surgery as treatment. A total of 12 preoperative variables were used with additional procedure matching: bilateral mastectomy, nodal surgery, tissue, and/or implant. Outcomes examined were 4 wound occurrences, sepsis, and unplanned return to the operating room. RESULTS: We identified 31,130 patient procedures with 2488 (7.5%) receiving chemotherapy. We matched 2411 cases, with probability of treatment being 0.005 to 0.470 in both cohorts. Superficial wound complication was the most common wound event, 2.24% in neoadjuvant-treated versus 2.45% in those that were not (P = 0.627). The rate of return to the operating room was 5.7% in the neoadjuvant group versus 5.2% in those that were not (P = 0.445). The rate of sepsis was 0.37% in the neoadjuvant group versus 0.46% in those that were not (P = 0.654). CONCLUSIONS: This large, matched cohort study, controlled for preoperative risk factors and most importantly for the surgical procedure performed, demonstrates that breast cancer patients receiving neoadjuvant chemotherapy have no increased risk for surgical morbidity.

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BACKGROUND: Previously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer. METHODS: The efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR. RESULTS: Immunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells. CONCLUSION: This is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors.

Factors associated with poor quality of life among cervical cancer survivors: implications for clinical care and clinical trials.

INTRODUCTION: The purpose of this study is to identify factors that are associated with poor quality of life (QOL) among cervical cancer survivors. METHODS: Patients identified through the California Cancer Registry were recruited to participate in a randomized counseling intervention. Patient-reported outcomes (PROs) were collected at study baseline (9-30 months post-diagnosis) and subsequent to the intervention. Multivariable linear models were used to identify independent factors associated with poor baseline QOL. RESULTS: Non-Hispanic (N=121) and Hispanic (N=83) women aged 22-73 completed baseline measures. Approximately 50% of participants received radiation therapy with or without chemotherapy. Compared to the US population, cervical cancer patients reported lower QOL and significantly higher levels of depression and anxiety (26% and 28% >1 SD above the general population means, respectively). Among those in the lowest quartile for QOL, 63% had depression levels >1 SD above the mean. In addition, treatment with radiation±chemotherapy (p=0.014), and self-reported comorbidities predating the cancer diagnosis (p<0.001) were associated with lower QOL. Sociodemographic characteristics explained only a small portion of variance in QOL (r(2)=0.23). Persistent gynecologic problems, low social support, depression, somatization, less adaptive coping, comorbidities, sleep problems and low education were all independently associated with low QOL in multivariate analysis (r(2)=0.74). CONCLUSION: We have identified key psychological and physical health factors that contribute significantly to poor quality of life subsequent to definitive cancer treatment. The majority of these factors are amenable to supportive care interventions and should be evaluated at the time of primary treatment.

Comparison of intraoperative versus delayed enteral feeding tube placement in patients undergoing a Whipple procedure.

BACKGROUND: The intraoperative placement of an enteral feeding tube (FT) during pancreaticoduodenectomy (PD) is based on the surgeon's perception of need for postoperative nutrition. Published preoperative risk factors predicting postoperative morbidity may be used to predict FT need and associated intraoperative placement. METHODS: A retrospective review of patients who underwent PD during 2005-2011 was performed by querying the National Surgical Quality Improvement Program (NSQIP) database with specific procedure codes. Patients were categorized based on how many of 10 possible preoperative risk factors they demonstrated. Groups of patients with scores of ≤ 1 (low) and ≥ 2 (high), respectively, were compared for FT need, length of stay (LoS) and organ space surgical site infections (SSIs). RESULTS: Of 138 PD patients, 82 did not have an FT placed intraoperatively, and, of those, 16 (19.5%) required delayed FT placement. High-risk patients were more likely to require a delayed FT (29.3%) compared with low-risk patients (9.8%) (P = 0.026). The 16 patients who required a delayed FT had a median LoS of 15.5 days, whereas the 66 patients who did not require an FT had a median LoS of 8 days (P < 0.001). CONCLUSIONS: In this analysis, subjects considered as high-risk patients were more likely to require an FT than low-risk patients. Assessment of preoperative risk factors may improve decision making for selective intraoperative FT placement.

Epigenetic memory emerging from integrated transcription bursts.

Some autonomous bacteria coordinate their actions using quorum-sensing (QS) signals to affect gene expression. However, noise in the gene environment can compromise the cellular response. By exercising precise control over a cell's genes and its microenvironment, we have studied the key positive autoregulation element by which the lux QS system integrates noisy signals into an epigenetic memory. We observed transcriptional bursting of the lux receptor in cells stimulated by near-threshold levels of QS ligand. The bursts are integrated over time into an epigenetic memory that confers enhanced sensitivity to the ligand. An emergent property of the system is manifested in pattern formation among phenotypes within a chemical gradient.

Evaluating Anti-tumor Immune Responses of Protein Nanoparticle-Based Cancer Vaccines.

Cancer vaccines displaying tumor-associated antigens (TAAs) train the immune system for enhanced tumor recognition and elimination. Nanoparticle-based cancer vaccines are ingested and processed by dendritic cells, which subsequently activate antigen-specific cytotoxic T cells, allowing them to identify and eliminate tumor cells displaying these TAAs. Here, we describe the procedures to conjugate TAA and adjuvant to a model protein nanoparticle platform (E2), followed by assessment of vaccine performance. Utilizing a syngeneic tumor model, the efficacy of in vivo immunization was determined by cytotoxic T lymphocyte assays and IFN-γ ELISpot ex vivo assays to measure tumor cell lysis and TAA-specific activation, respectively. In vivo tumor challenge directly allows evaluation of anti-tumor response and survival over time.

Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display.

Cancer vaccine immunotherapy facilitates the immune system's recognition of tumor-associated antigens, and the biomolecular design of these vaccines using nanoparticles is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed towards vaccine efficacy, participation by highly cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We examined vaccines based on the E2 protein nanoparticle platform that delivered encapsulated TLR9 agonist bacterial-like DNA (CpG1826 or CpG1018) or TLR7 agonist viral ssRNA to determine their efficacy over free agonists in activating both mDCs and pDCs for antigen presentation. Although mDCs were only activated by nanoparticle-encapsulated TLR9 agonists, pDCs were activated by all the individually tested constructs, and CpG1826 was shown to induce pDC cytokine production. Transfer of secreted factors from pDCs that were stimulated with a vaccine formulation comprising peptide antigen and CpG1826 enhanced mDC display of the antigen, particularly when delivered in nanoparticles. Only when treated with nanoparticle-conjugated vaccine could pDCs secrete factors to induce antigen display on naïve mDCs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing effective cancer vaccines, and demonstrate the advantage of using nanoparticle-based vaccine delivery.

Granulocyte-macrophage stimulating factor (GM-CSF) increases circulating dendritic cells but does not abrogate suppression of adaptive cellular immunity in patients with metastatic colorectal cancer receiving chemotherapy.

BACKGROUND: Advanced cancer and chemotherapy are both associated with immune system suppression. We initiated a clinical trial in patients receiving chemotherapy for metastatic colorectal cancer to determine if administration of GM-CSF in this setting was immunostimulatory. METHODS: Between June, 2003 and January, 2007, 20 patients were enrolled in a clinical trial (NCT00257322) in which they received 500 ug GM-CSF daily for 4 days starting 24 hours after each chemotherapy cycle. There were no toxicities or adverse events reported. Blood was obtained before chemotherapy/GM-CSF administration and 24 hours following the final dose of GM-CSF and evaluated for circulating dendritic cells and adaptive immune cellular subsets by flow cytometry. Peripheral blood mononuclear cell (PBMC) expression of γ-interferon and T-bet transcription factor (Tbx21) by quantitative real-time PCR was performed as a measure of Th1 adaptive cellular immunity. Pre- and post-treatment (i.e., chemotherapy and GM-CSF) samples were evaluable for 16 patients, ranging from 1 to 5 cycles (median 3 cycles, 6 biologic sample time points). Dendritic cells were defined as lineage (-) and MHC class II high (+). RESULTS: 73% of patients had significant increases in circulating dendritic cells of ~3x for the overall group (5.8% to 13.6%, p = 0.02) and ~5x excluding non-responders (3.2% to 14.5%, p < 0.001). This effect was sustained over multiple cycles for approximately half of the responders, but tachyphylaxis over subsequent chemotherapy cycles was noted for the remainder. Treatment also led to a significant reduction in the proportion of circulating regulatory T-cells (Treg; p = 0.0042). PBMC Tbx21 levels declined by 75% following each chemotherapy cycle despite administration of GM-CSF (p = 0.02). PBMC γ-interferon expression, however was unchanged. CONCLUSIONS: This clinical trial confirms the suppressive effects of chemotherapy on Th1 cellular immunity in patients with metastatic colorectal cancer but demonstrates that mid-cycle administration of GM-CSF can significantly increase the proportion of circulating dendritic cells. As the role of dendritic cells in anti-tumor immunity becomes better defined, GM-CSF administration may provide a non-toxic intervention to augment this arm of the immune system for cancer patients receiving cytotoxic therapy. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00257322.

Using a nanopore for single molecule detection and single cell transfection.

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.