Targeting interleukin-6 as a treatment approach for peritoneal carcinomatosis.

Peritoneal carcinomatosis (PC) is a complex manifestation of abdominal cancers, with a poor prognosis and limited treatment options. Recent work identifying high concentrations of the cytokine interleukin-6 (IL-6) and its soluble receptor (sIL-6-Rα) in the peritoneal cavity of patients with PC has highlighted this pathway as an emerging potential therapeutic target. This review article provides a comprehensive overview of the current understanding of the potential role of IL-6 in the development and progression of PC. We discuss mechansims by which the IL-6 pathway may contribute to peritoneal tumor dissemination, mesothelial adhesion and invasion, stromal invasion and proliferation, and immune response modulation. Finally, we review the prospects for targeting the IL-6 pathway in the treatment of PC, focusing on common sites of origin, including ovarian, gastric, pancreatic, colorectal and appendiceal cancer, and mesothelioma.

Characterizing the Immune Environment in Peritoneal Carcinomatosis: Insights for Novel Immunotherapy Strategies.

BACKGROUND OR PURPOSE: Carcinomatosis, a distinct pattern of metastatic cancer in the peritoneal cavity, poses challenges for treatment and has limited therapeutic options. Understanding the immune environment of peritoneal surface malignancies is crucial for developing effective immunotherapeutic approaches. This study characterizes soluble immune mediators in the peritoneal fluid of patients with and without carcinomatosis to identify targets for novel treatment strategies. PATIENTS AND METHODS: Serum and peritoneal fluid samples were collected from surgical patients, and a multianalyte analysis was performed using the Luminex platform. Patient characteristics, tumor sites, and sample collection details were recorded. Soluble immune mediator levels were measured and compared between peritoneal fluid and serum samples and among clinical subgroups. Statistical analysis was conducted to assess differences in analyte concentrations and correlations between samples. RESULTS: There were 39 patients included in the study, with varying surgical indications. Significant differences were observed in soluble immune mediator levels between peritoneal fluid and serum, with peritoneal fluid exhibiting lower concentrations. Carcinomatosis was associated with elevated levels of proinflammatory mediators, including IL-6 and IL-8, while adaptive immune response markers were low in peritoneal fluid. CONCLUSIONS: The peritoneal immune microenvironment in carcinomatosis favors innate immunity, presenting a challenging environment for effective antitumor response. High levels of proinflammatory mediators suggest potential targets for intervention, such as the IL-6 axis, FGF2, IL-8, and CCL2; these could be explored as potential mitigators of malignant ascites and enhance anti-tumor immune responses. These findings provide valuable insights for developing immunotherapy strategies and improving outcomes in patients with peritoneal carcinomatosis.

Predicting the CD34 content of mobilized peripheral blood leukapheresis products: single institution experience over 20 years.

BACKGROUND AIMS: Since the standardization of CD34 measurement by flow cytometry, predictors of leukapheresis CD34 yield have played a pivotal role in planning donor leukaphereses. We describe here a single institution's experience with a multivariate predictor that was used for 2,929 products without alteration for 20 years. METHODS: The ordinary least squares regression model variables included log peripheral CD34 count, collection duration (3- versus 4-hours), collection number, donor sex, and transplant type. RESULTS: During the study period we changed flow cytometers twice and leukapheresis instruments once. During the Cobe Spectra era the predictor explained 90% of the variability in CD34 collection yield for autologous transplants (r2 = 0.90), and 70% for allogeneic transplants with an overall sensitivity to predict a CD34 yield of ≥ 1 × 106/kg of 97.7%, and specificity of 81.4%. CONCLUSIONS: Implemented prospectively with real-time result reporting, the model allowed us to predict CD34 yield with both 3- and 4-hour collection scenarios. Given this guidance, 3-hour collections were selected by the clinical team 25% of the time, saving patient leukapheresis time and resources. When faced with a prediction of < 1 × 106 CD34/kg, the clinical team chose to defer collection 72% of the time. In instances where leukapheresis was performed despite a poor predicted outcome, 85% of patients collected on the Cobe Spectra, and 92% of patients collected on the Optia, failed to collect at least 1 × 106 CD34/kg. A revised model is tested retrospectively on Optia data, and suggestions for further improvements are discussed.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

BACKGROUND: Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). STUDY DESIGN AND METHODS: Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N = 4 healthy adult volunteers). One bag was biotinylated (15 µg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3 µg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). RESULTS: Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2  = 15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. CONCLUSION: Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Changing the Paradigm of Craniofacial Reconstruction: A Prospective Clinical Trial of Autologous Fat Transfer for Craniofacial Deformities.

OBJECTIVE: This study aimed to prospectively assess outcomes for surgical autologous fat transfer (AFT) applied for traumatic and postsurgical craniofacial deformities. The minimally invasive nature of AFT has potential for reduced risk and superior outcomes compared with current reconstructive options. BACKGROUND: Craniofacial deformities have functional and psychosocial sequelae and can profoundly affect quality of life. Traditional reconstructive options are invasive, invasive, complex, and often lack precision in outcomes. Although AFT is safe, effective, and minimally invasive, only anecdotal evidence exists for reconstruction of craniofacial deformities. METHODS: In this Institutional Review Board-approved prospective cohort study, 20 subjects underwent AFT (average volume: 23.9 ±â€Š13.2 mL). Volume retention over time was determined using high-resolution computed tomography. Flow cytometry was used to assess cellular subpopulations and viability in the stromal vascular fraction. Quality of life assessments were performed. After the completion of 9-month follow-up, 5 subjects were enrolled for a second treatment. RESULTS: No serious adverse events occurred. Volume retention averaged 63 ±â€Š17% at 9 months. Three-month retention strongly predicted 9-month retention (r=0.996, P < 0.0001). There was no correlation between the total volume injected and retention. Patients undergoing a second procedure had similar volume retention as the first (P = 0.05). Age, sex, body mass index, and stromal vascular fraction cellular composition did not impact retention. Surprisingly, former smokers had greater volume retention at 9 months compared with nonsmokers (74.4% vs 56.2%, P = 0.009). Satisfaction with physical appearance (P = 0.002), social relationships (P = 0.02), and social functioning quality of life (P = 0.05) improved from baseline to 9 months. CONCLUSIONS: For craniofacial defects, AFT is less invasive and safer than traditional reconstructive options. It is effective, predictable, and reaches volume stability at 3 months. Patient-reported outcomes demonstrate a positive life-changing impact.

Evaluation of Exosome Proteins by on-Bead Flow Cytometry.

Exosomes, recently re-named "small extracellular vesicles" or "sEV," are emerging as an intercellular communication system. Quantification of the molecular cargo exosomes carry by on-bead flow cytometry is needed for defining their role in information transfer and in human disease. Exosomes (sEV) isolated from cell supernatants or plasma of cancer patients by size-exclusion chromatography were captured by biotinylated antibodies specific for antigens in the exosome cargo (e.g., tetraspanins) and placed on streptavidin-labeled beads. Detection was performed with pretitered fluorochrome-labeled antibodies of desired specificity. The data were acquired in a conventional cytometer, and molecules of equivalent soluble fluorochrome (MESF) beads were used to quantify the number of fluorescent molecules bound per bead. Isotype antibody controls were obligatory. The mean fluorescence intensity (MFI) value of each sample was converted into MESF units, and the separation index (SI), which quantifies separation of stained and isotype control beads, was determined. Various proteins identified by labeled antibodies were quantified on the surface of tumor cell-derived exosomes. To identify intravesicular cargo, such as cytokines or chemokines, exosomes were lysed with 0.3% Triton-100, and the proteins in lysates were loaded on aldehyde/sulfate latex beads for flow cytometry. Examples of quantitative surface and/or intravesicular on-bead flow cytometry for exosomes produced by various cells or present in body fluids of cancer patients are provided. On-bead flow cytometry standardized for use with conventional cytometers is a useful method for protein detection and quantitation in exosomes isolated from supernatants of cell lines or plasma of patients with cancer. © 2020 International Society for Advancement of Cytometry.

Current good manufacturing practices-compliant manufacture and measurement of biotin-labeled red blood cells.

BACKGROUND: Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. METHODS: We have developed a closed-system, current good manufacturing practices (cGMP)-compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. RESULTS: The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin-labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. DISCUSSION: We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.

Improved quantitative detection of biotin-labeled red blood cells by flow cytometry.

BACKGROUND: Biotin-labeled red blood cells (BioRBC) can be tracked after transfusion, providing a convenient and safe way to measure RBC survival in vivo. RBC survival is of interest for determining optimal blood storage conditions and for assessing the impact of genetic and biologic variants in blood donors on the survival of transfused RBCs. Here we present an improved, platform-independent assay for quantifying biotin on BioRBC. This approach is also useful for detecting BioRBC in peripheral blood samples as rare events. STUDY DESIGN AND METHODS: We optimized the signal-to-noise ratio of the detecting reagent (phycoerythrin-conjugated streptavidin [SA-PE]) by determining the SA-PE concentration yielding the greatest separation index between BioRBC and unlabeled RBCs. We calibrated the fluorescence intensity measurements to molecules of equivalent soluble fluorochrome (MESF), a quantitative metric of fluorochrome binding and therefore of biotin bound per RBC. We then characterized the limit of blank and limit of quantification (LoQ) for BioRBC labeled at different densities. RESULTS: Biotin-labeled RBCs at sulfo-NHS-biotin concentrations of 3 to 30 µg/mL (27-271 nmol/mL RBCs) ranged from approximately 32,000 to 200,000 MESF/RBC. The LoQ ranged from one in 274,000 to one in 649,000, depending on biotin-labeling density. CONCLUSION: Increased sensitivity to detect BioRBC may facilitate tracking over longer periods and/or reduction of the BioRBC dose. Total RBC-bound biotin dose has been shown to correlate with the likelihood of developing antibodies to BioRBC. Lowering the dose of labeled cells may help avoid this eventuality.

Proteomic Profiling of Native Unpassaged and Culture-Expanded Mesenchymal Stromal Cells (MSC).

Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.

Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment.

Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.

Antibody-based cell-surface proteome profiling of metastatic breast cancer primary explants and cell lines.

Flow cytometric cell surface proteomics provides a new and powerful tool to determine changes accompanying neoplastic transformation and invasion, providing clues to essential interactions with the microenvironment as well as leads for potential therapeutic targets. One of the most important advantages of flow cytometric cell surface proteomics is that it can be performed on living cells that can be sorted for further characterization and functional studies. Here, we document the surface proteome of clonogenic metastatic breast cancer (MBrCa) explants, which was strikingly similar to that of normal mesenchymal stromal cells (P = 0.017, associated with Pearson correlation coefficient) and transformed mammary epithelial cells (P = 0.022). Markers specifically upregulated on MBrCa included CD200 (Ox2), CD51/CD61 (Integrin α5/ß3), CD26 (dipeptidyl peptidase-4), CD165 (c-Cbl), and CD54 (ICAM-1). Proteins progressively upregulated in a model of neoplastic transformation and invasion included CD26, CD63 (LAMP3), CD105 (Endoglin), CD107a (LAMP1), CD108 (Semaphorin 7A), CD109 (Integrin ß4), CD151 (Raph blood group), and disialoganglioside G2. The proteome of the commonly used cell lines MDA-MB-231, MCF7, and BT-474 were uncorrelated with that of MBrCa (P = 1.0, 1.0, 0.9, respectively). The comparison has demonstrated the mesenchymal nature of clonogenic cells isolated by short-term culture of metastatic breast cancer, provided several leads for biomarkers and potential targets for anti-invasive therapy, including CD200, and highlighted the limitations of breast cancer cell lines for representing the cell surface biology of breast cancer. © 2017 International Society for Advancement of Cytometry.

BOK displays cell death-independent tumor suppressor activity in non-small-cell lung carcinoma.

As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFß-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5.

The use of simultaneous confidence bands for comparison of single parameter fluorescent intensity data.

Despite the utility of multiparameter flow cytometry for a wide variety of biological applications, comparing single parameter histograms of fluorescence intensity remains a mainstay of flow cytometric analysis. Even comparisons requiring multiparameter gating strategies often end with single parameter histograms as the final readout. When histograms overlap, analysis relies on comparison of mean or median fluorescence intensities, or determination of percent positive based on an arbitrary cutoff. Earlier attempts to address this problem utilized either simple channel-by-channel subtraction without statistical evaluation, or the Kolmogorov-Smirnov (KS) or Chi-square test statistics, both of which proved to be overly sensitive to small and biologically insignificant differences. Here we present a method for the comparison of two single-parameter histograms based on difference curves and their simultaneous confidence bands generated by bootstrapping raw channel data. Bootstrapping is a nonparametric statistical approach that can be used to generate confidence intervals without distributional assumptions about the data. We have constructed simultaneous confidence bands and show them to be superior to KS and Cox methods. The method constructs 95% confidence bands about the difference curves, provides a P value for the comparison and calculates the area under the difference curve (AUC) as an estimate of percent positive and the area under the confidence band (AUCSCB95), providing a lower estimate of the percent positive. To demonstrate the utility of this new approach we have examined single-color fluorescence intensity data taken from a cell surface proteomic survey of a lung cancer cell line (A549) and a published fluorescence intensity data from a rhodamine efflux assay of P-glycoprotein activity, comparing rhodamine 123 loading and efflux in CD4 and CD8 T-cell populations. SAS source code is provided as supplementary material.

A depleting antibody toward sca-1 mitigates a surge of CD34(+)/c-kit(+) progenitors and reduces vascular restenosis in a murine vascular injury model.

OBJECTIVE: Vascular restenosis remains a major obstacle to long-term success after vascular intervention. Circulating progenitor cells have been implicated in restenosis, and yet it has remained unclear if these cells, particularly nonendothelial progenitors, have an active role in this pathologic process. We hypothesized that circulating CD34(+)/c-kit(+) progenitors would increase after vascular injury, mirrored by changes in the injury signal, stromal cell-derived factor 1α (sdf1α). We further postulated that an antibody-based depletion would mitigate progenitor surge and, in turn, reduce restenosis in a murine model. METHODS: C57BL6 mice underwent wire injury of the femoral artery and were compared with mice with sham surgery and vessel ligation by flow cytometry as well as by sdf1α enzyme-linked immunosorbent assay of peripheral blood. Next, injured C57BL6 mice treated with a depleting antibody toward the progenitor marker sca-1 or with an isotype control were compared in terms of sdf1α as well as enumeration of progenitors. At 28 days, restenosis was quantified between sca-1- and isotype-treated animals. RESULTS: Wire injury generated an increase in sdf1α as well as a surge of CD34(+)/c-kit(+) progenitors relative to nonsurgical controls (P = .005). Treatment with sca-1 antibody ablated the peripheral surge compared with isotype-treated, injured animals (P = .02), and sca progenitor depletion reduced the 28-day intima to media ratio in a statistically significant fashion compared with either nontreated (P = .04) or isotype-treated (P = .036) animals. CONCLUSIONS: Our study has demonstrated that sca-1 antibody reduces both progenitor surge and vascular restenosis after endoluminal vascular injury in a murine model. This suggests that circulating progenitors play an active role in restenotic disease.

Coping with artifact in the analysis of flow cytometric data.

This article highlights several sources of artifact that interfere with optimal analysis of flow cytometric data. Such problems are compounded when flow cytometry is performed on mechanically and enzymatically disaggregated solid tissues or on cultured cells, where subcellular debris, apoptotic or necrotic cells, and highly autofluorescent cells may comprise a substantial proportion of acquired events. We provide real-world examples of tissues that pose specific analytical challenges (bone marrow, breast cancer, lung cancer and adipose tissue) and suggest approaches to improve data analysis. These include the use of a sequential or hierarchical gating process, which envisions analysis as consisting of three parts: (1) removal of artifact; (2) defining classifying populations; and (3) measuring outcomes on the classifying populations. Tools for removal of artifact include use of the time parameter to detect and remove fluidic perturbations, use of doublet discrimination to avoid analysis of cell clusters, measurement of DNA content to remove subcellular debris and late apoptotic cells, Boolean gating to recognize and remove auto-fluorescent events, and the use of a dump gate (markers known to be negative on the population of interest, but expressed on interfering cells). Implementation of these strategies, as appropriate, extends the usefulness of flow cytometry to a wider range of applications.

The cell-surface proteome of cultured adipose stromal cells.

In this technical note we describe a method to evaluate the cell surface proteome of human primary cell cultures and cell lines. The method utilizes the BD Biosciences lyoplate, a system covering 242 surface proteins, glycoproteins, and glycosphingolipids plus relevant isotype controls, automated plate-based flow cytometry, conventional file-level analysis and unsupervised K-means clustering of markers on the basis of percent of positive events and mean fluorescence intensity of positive and total clean events. As an example, we determined the cell surface proteome of cultured adipose stromal cells (ASC) derived from 5 independent clinical isolates. Between-sample agreement of very strongly expressed (n = 32) and strongly expressed (n =16) markers was excellent, constituting a reliable profile for ASC identification and determination of functional properties. Known mesenchymal markers (CD29, CD44, CD73, CD90, CD105) were among the identified strongly expressed determinants. Among other strongly expressed markers are several that are potentially immunomodulatory including three proteins that protect from complement mediated effects (CD46, CD55, and CD59), two that regulate apoptosis (CD77 and CD95) and several with ectoenzymatic (CD10, CD26, CD13, CD73, and CD143) or receptor tyrosine kinase (CD140b (PDGFR), CD340 (Her-2), EGFR) activity, suggesting mechanisms for the anti-inflammatory and tissue remodeling properties of ASC. Because variables are standardized for K-means clustering, results generated using this methodology should be comparable between instrumentation platforms. It is widely generalizable to human primary explant cultures and cells lines and will prove useful to determine how cell passage, culture interventions, and gene expression and silencing affect the cell-surface proteome.

CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury.

BACKGROUND: Intimal hyperplasia (restenosis) is an exaggerated healing response leading to failure of half of vascular interventions. Increasing evidence suggests that circulating progenitor cells contribute to intimal pathology, and clinical studies have demonstrated a correlation between progenitor cells and the incidence of restenosis after cardiovascular interventions. The aims of this study were to characterize the temporal response of CD34+ progenitors following vascular injury in an ovine model and to evaluate an affinity pheresis approach to attenuate this response. METHODS: An ovine model underwent either operative vascular injury or a nonvascular surgery (n = 3 per group). Blood was examined perioperatively over 2 weeks by flow cytometry. Next, an affinity pheresis approach to mediate systemic depletion of CD34 progenitors was designed. Custom agarose pheresis matrix with antibody affinity toward CD34 or an isotype control was evaluated in vitro. Next, following vascular injury, sheep underwent perioperative whole blood volume pheresis toward either the progenitor cell marker CD34 (n = 3) or an isotype control (n = 4) for 14 days. Animals were monitored by physical exam as well as complete blood counts. Cells recovered by pheresis were eluted and examined by flow cytometry. RESULTS: Flow cytometry revealed a focal surge of circulating CD34 cells after vascular injury but not among surgical controls (P = .05). Toward the goal of an approach to attenuate the surge of CD34 progenitors, an evaluation of high-flow affinity matrix revealed efficacy in removal of progenitors from ovine blood in vitro. Next, a separate group of animals undergoing affinity pheresis after vascular injury was evaluated to mediate systemic depletion of CD34+ cells. Again, a surge of CD34+ cells was observed among isotype pheresis animals following vascular intervention but was attenuated over 20-fold by a CD34 pheresis approach (P = .029). Furthermore, an average of 77 million CD34-positive cells were eluted from the CD34 pheresis matrix. Despite multiple sessions of pheresis, complete blood counts remained essentially unchanged over 2 weeks. CONCLUSIONS: Despite evidence suggesting a role for CD34+ circulating progenitor cells in restenotic pathology, the temporal pattern of CD34 progenitors after vascular injury has not been previously defined. We have demonstrated a surge among circulating CD34+ cells that appears confined to procedures involving vascular injury and that this event seems to occur early after vascular injury. We further conclude that CD34 affinity pheresis attenuates the surge. This approach for direct depletion of progenitors may have important implications for the study of progenitors in vascular restenosis.

Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.

OBJECTIVE: To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. BACKGROUND: Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol"). METHODS: Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring. RESULTS: All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates. CONCLUSIONS: Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Statistical classification of multivariate flow cytometry data analyzed by manual gating: stem, progenitor, and epithelial marker expression in nonsmall cell lung cancer and normal lung.

The use of supervised classification to extract markers from primary flow cytometry data is an emerging field that has made significant progress, spurred by the growing complexity of multidimensional flow cytometry. Whether the markers are extracted without supervision or by conventional gate and region methods, the number of candidate variables identified is typically larger than the number of specimens (p > n) and many variables are highly intercorrelated. Thus, comparison across groups or treatments to determine which markers are significant is challenging. Here, we utilized a data set in which 86 variables were created by conventional manual analysis of individual listmode data files, and compared the application of five multivariate classification methods to discern subtle differences between the stem/progenitor content of 35 nonsmall cell lung cancer and adjacent normal lung specimens. The methods compared include elastic-net, lasso, random forest, diagonal linear discriminant analysis, and best single variable (best-1). We described a broadly applicable methodology consisting of: 1) variable transformation and standardization; 2) visualization and assessment of correlation between variables; 3) selection of significant variables and modeling; and 4) characterization of the quality and stability of the model. The analysis yielded both validating results (tumors are aneuploid and have higher light scatter properties than normal lung), as well as leads that require followup: Cytokeratin+ CD133+ progenitors are present in normal lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more prevalent in tumor. We anticipate that the methods described here will be broadly applicable to a variety of multidimensional cytometry problems.