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.

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.

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.

Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging.

Bioluminescence imaging has advantages over fluorescence imaging, such as minimal photobleaching and autofluorescence, and greater signal-to-noise ratios in many complex environments. Although significant achievements have been made in luciferase engineering for generating bright and stable reporters, the full capability of luciferases for nanoparticle tracking has not been comprehensively examined. In biocatalysis, enhanced enzyme performance after immobilization on nanoparticles has been reported. Thus, we hypothesized that by assembling luciferases onto a nanoparticle, the resulting complex could lead to substantially improved imaging properties. Using a modular bioconjugation strategy, we attached NanoLuc (NLuc) or Akaluc bioluminescent proteins to a protein nanoparticle platform (E2), yielding nanoparticles NLuc-E2 and Akaluc-E2, both with diameters of ∼45 â€‹nm. Although no significant differences were observed between different conditions involving Akaluc and Akaluc-E2, free NLuc at pH 5.0 showed significantly lower emission values than free NLuc at pH 7.4. Interestingly, NLuc immobilization on E2 nanoparticles (NLuc-E2) emitted increased luminescence at pH 7.4, and at pH 5.0 showed over two orders of magnitude (>200-fold) higher luminescence (than free NLuc), expanding the potential for imaging detection using the nanoparticle even upon endocytic uptake. After uptake by macrophages, the resulting luminescence with NLuc-E2 nanoparticles was up to 7-fold higher than with free NLuc at 48 â€‹h. Cells incubated with NLuc-E2 could also be imaged using live bioluminescence microscopy. Finally, biodistribution of nanoparticles into lymph nodes was detected through imaging using NLuc-E2, but not with conventionally-labeled fluorescent E2. Our data demonstrate that NLuc-bound nanoparticles have advantageous properties that can be utilized in applications ranging from single-cell imaging to in vivo biodistribution.

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.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

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.

"I need to have a fulfilling job": A qualitative study of surgeon well-being and professional fulfillment.

BACKGROUND: Burnout, often regarded as an individual failing, rather than a systemic one, negatively impacts quality of care, patient safety and healthcare costs. Focusing on improving well-being can help mitigate burnout. This study examined protective factors that promote well-being and professional fulfillment in surgeons. METHODS: Using a purposive sample, 32 semi-structured 30-60-min interviews were conducted with surgeons of varying sub-specialties and rank. Abductive exploratory analysis was used to code and interpret interview transcripts and to build a conceptual model of surgeon well-being. RESULTS: Emergent protective factors were placed into one of three levels of implementation: individual, team-level, and institutional (figure). Individual factors for well-being included autonomy and adequate time to pursue non-clinical endeavors. Team-level factors consisted of adaptability, boundaries, and cohesion. Institutional factors related to diversifying performance evaluations and celebrating and recognizing individual value and contributions. CONCLUSIONS: The conceptual model developed from the results of this study highlights factors important to surgeons' professional well-being. This model can be used to guide quality improvement efforts.

Preoperative multimodal protocol reduced postoperative nausea and vomiting in patients undergoing mastectomy with reconstruction.

BACKGROUND: Mastectomy with immediate reconstruction is a high-risk cohort for postoperative nausea and vomiting (PONV). Known risk factors for PONV include female gender, prior PONV history, nonsmoker, age < 50, and postoperative opioid exposure. The objective of this observational, cohort analysis was to determine whether a standardized preoperative protocol with nonopioid and anti-nausea multimodal medications would reduce the odds of PONV. METHODS: After IRB approval, retrospective data were collected for patients undergoing mastectomy with or without a nodal resection, and immediate subpectoral tissue expander or implant reconstruction. Patients were grouped based on treatment: those receiving the protocol - oral acetaminophen, pregabalin, celecoxib, and transdermal scopolamine (APCS); those receiving none (NONE), and those receiving partial protocol (OTHER). Logistic regression models were used to compare PONV among treatment groups, adjusting for patient and procedural variables. MAIN FINDINGS: Among 305 cases, the mean age was 47 years (21-74), with 64% undergoing a bilateral procedure and 85% having had a concomitant nodal procedure. A total of 44.6% received APCS, 30.8% received OTHER, and 24.6% received NONE. The APCS group had the lowest rate of PONV (40%), followed by OTHER (47%), and NONE (59%). Adjusting for known preoperative variables, the odds of PONV were significantly lower in the APCS group versus the NONE group (OR=0.42, 95% CI: 0.20, 0.88 p = 0.016). CONCLUSIONS: Premedication with a relatively inexpensive combination of oral non-opioids and an anti-nausea medication was associated with a significant reduction in PONV in a high-risk cohort. Use of a standardized protocol can lead to improved care while optimizing the patient experience.

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.

Benchmarks for Academic Oncology Faculty.

The role of clinical researchers is vital to cancer progress. The teaching, research, and leadership roles that academic oncologists hold need to be accounted for and appropriately compensated. National metrics are currently inexistent, but are necessary to move the oncology research field forward. Clinical research and routine clinical care must be harmoniously integrated without competing. This article reviews the national landscape of clinical cancer research and proposes a call for action.

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.

An Antigen-Delivery Protein Nanoparticle Combined with Anti-PD-1 Checkpoint Inhibitor Has Curative Efficacy in an Aggressive Melanoma Model.

Immune checkpoint inhibition is a promising alternative treatment to standard chemotherapies; however, it fails to achieve long-term remission in a significant portion of patients. A previously developed protein nanoparticle-based platform (E2 nanoparticle) delivers cancer antigens to increase antigen-specific tumor responses. While prior work has focussed on prophylactic conditions, the objectives in this study are therapeutic. It is hypothesized that immune checkpoint inhibition, when augmented by antigen delivery using E2 nanoparticles containing CpG oligonucleotide 1826 (CpG) and a glycoprotein 100 (gp100) melanoma antigen epitope (CpG-gp-E2), would synergistically elicit antitumor responses. To identify a regimen primed for obtaining effective treatment results, immune benchmarks in the spleen and tumor are examined. Conditions that lead to significant immune activation, including increases in gp100-specific interferon gamma (IFN-𝜸), CD8 T cells in the spleen, tumor-infiltrating CD8 T cells, and survival time are identified. Based on the findings, the resulting combination of CpG-gp-E2 and anti-programmed cell death protein 1 (anti-PD-1) treatment in tumor-challenged mice yield significantly increased long-term survival; more than 50% of the mice treated with combination therapy were tumor-free, compared with 0% and ≈5% for CpG-gp-E2 and anti-PD-1 alone, respectively. Evidence of a durable antitumor response is also observed upon tumor rechallenge, pointing to long-lasting immune memory.

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.

Perioperative antibiotics should be used for placement of implanted central venous ports: A propensity analysis evaluating risk.

OBJECTIVE: To quantify risk for CRI based on PABX use in CVAP placement for cancer patients. SUMMARY BACKGROUND DATA: Central venous access ports (CVAP) are totally implanted devices used for chemotherapy. There is a temporal risk for catheter related infection (CRI) to insertion and perioperative prophylactic antibiotics (PABX) use is a contested issue among practitioners. METHODS: Data was collected from a single center, academic oncology center. Treatment with a perioperative PABX was compared to non-treatment, to examine the incidence of 14-day CRI. Propensity scores with matched weights controlled for confounding, using 15 demographic, procedural and clinical variables. RESULTS: From 2007 to 2012, 1,091 CVAP were placed, where 59.7 % received PABX. The 14-day CRI rate was 0.82%, with 78% of those not receiving PABX. While results did not achieve statistical significance, use of PABX was associated with a 58% reduction in the odds of a 14-day CRI (OR = 0.42, 95% CI: 0.08-2.24, p = 0.31). CONCLUSION: The findings suggest a reduction in early CRI with the use of PABX. Since CRI treatment can range from a course of oral antibiotics, port removal, to hospital admission, we suggest clinicians consider these data when considering PABX in this high-risk population.

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.

Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells.

Tissues are increasingly being analyzed at the single cell level in order to characterize cellular diversity and identify rare cell types. Single cell analysis efforts are greatly limited, however, by the need to first break down tissues into single cell suspensions. Current dissociation methods are inefficient, leaving a significant portion of the tissue as aggregates that are filtered away or left to confound results. Here, we present a simple and inexpensive microfluidic device that simultaneously filters large tissue fragments and dissociates smaller aggregates into single cells, thereby improving single cell yield and purity. The device incorporates two nylon mesh membranes with well-defined, micron-sized pores that operate on aggregates of different size scales. We also designed the device so that the first filtration could be performed under tangential flow to minimize clogging. Using cancer cell lines, we demonstrated that aggregates were effectively dissociated using high flow rates and pore sizes that were smaller than a single cell. However, pore sizes that were less than half the cell size caused significant damage. We then improved results by passing the sample through two filter devices in series, with single cell yield and purity predominantly determined by the pore size of the second membrane. Next, we optimized performance using minced and digested murine kidney tissue samples, and determined that the combination of 50 and 15 µm membranes was optimal. Finally, we integrated these two membranes into a single filter device and performed validation experiments using minced and digested murine kidney, liver, and mammary tumor tissue samples. The dual membrane microfluidic filter device increased single cell numbers by at least 3-fold for each tissue type, and in some cases by more than 10-fold. These results were obtained in minutes without affecting cell viability, and additional filtering would not be required prior to downstream applications. In future work, we will create complete tissue analysis platforms by integrating the dual membrane microfluidic filter device with additional upstream tissue processing technologies, as well as downstream operations such as cell sorting and detection.

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.