Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer.

BACKGROUND: The prognostic value of tumor-associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co-expressing the immunoinhibitory molecule PD-L1 and their spatial relationship with CD8+ T-cells in patients treated for stage III colon cancer. METHODS: Tissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD-L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan-Meier estimates and Cox regression were used to assess survival. RESULTS: Intratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33-.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33-.83, P = .006), intratumoral CD11c+ PD-L1+ cell density (HR = .57, 95% CI .35-.92, P = .021), and stromal CD11c+ PD-L1+ cell density (HR = .48, 95% CI .30-.77, P = .003) on leading-edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+ PD-L1+ cell density in tumor epithelium and stromal compartments. CONCLUSION: Here we showed that PD-L1-expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically "hot" tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor-associated DC may help to further elucidate their prognostic value.

An active, collaborative approach to learning skills in flow cytometry.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera.

An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze-thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators.

Receptor activator of NF-κB ligand promotes proliferation of a putative mammary stem cell unique to the lactating epithelium.

In mice, CD49f(hi) mammary stem cells (MaSCs) asymmetrically divide to generate CD49f(+) committed progenitor cells that differentiate into CD49f(-) phenotypes of the milk-secreting tissue at the onset of pregnancy. We show CD49f(+) primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy-associated hormones (PAH) compared with CD49f(+) cells from nonlactating tissue. Differentiation of CD49f(+) PMEC in extracellular matrix produces CD49f(-) luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f(-) luminal cells, while receptor activator of nuclear factor (NF)-κB ligand (RANKL) specifically stimulates division of basal CD49f(+) cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self-renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH.

The Prognostic and Predictive Value of SOX2+ Cell Densities in Patients Treated for Colorectal Cancer.

SOX2 (sex-determining region-Y homeobox-2) is a transcription factor essential for the maintenance of pluripotency and is also associated with stem-cell-like properties in preclinical cancer models. Our previous study on a cohort of stage III colon cancer patients demonstrated high SOX2+ cell densities were associated with poor prognosis. However, most patients were treated with adjuvant chemotherapy so the prognostic value of SOX2 could not be assessed independently from its value as a predictive marker for non-response to chemotherapy. This study aimed to assess whether SOX2 was a true prognostic marker or a marker for chemotherapy response in a historical cohort of patients, a high proportion of whom were chemotherapy-naïve. SOX2 immunostaining was performed on tissue micro-arrays containing tumor cores from 797 patients with stage II and III colorectal cancer. SOX2+ cell densities were then quantified with StrataQuest digital image analysis software. Overall survival was assessed using Kaplan-Meier estimates and Cox regression. It was found that high SOX2+ cell densities were not associated with poor overall survival. Furthermore, all patients had a significant improvement in survival after 5-fluorouracil (5-FU) treatment, irrespective of their SOX2+ cell density. Therefore, SOX2+ cell densities were not associated with prognosis or chemotherapy benefit in this study. This is in contrast to our previous study, in which most patients received oxaliplatin as part of their treatment, in addition to 5-FU. This suggests SOX2 may predict response to oxaliplatin treatment, but not 5-FU.

Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system.

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response.

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response.

T cell cytokine responses to stimulation with Ureaplasma parvum in pregnancy.

UNLABELLED: Ureaplasma spp. are a common vaginal microorganism causally linked to inflammation-driven preterm birth (PTB). The nature of the immune response to Ureaplasma spp. may influence PTB risk. This study sought to define maternal T cell cytokine responses to in vitro stimulation with Ureaplasma parvum serovar 3 (UpSV3) in vaginally colonised (UP+) and non-colonised (UP-) pregnant women. Whole blood flow cytometry demonstrated an increase (p=0.027) in the baseline frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells in UP+ women. UpSV3 stimulation resulted in a significant and specific increase (p=0.001) in the frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells, regardless of vaginal colonisation status. UpSV3 stimulation also increased the frequency of IFNγ-positive CD3(+)CD4(+) T cells, particularly in the UP+ group (p=0.003). This is the first published study to examine T cell responses to Ureaplasma spp. EXPOSURE: Future appropriately-powered studies are needed to assess whether insufficient priming or a loss of tolerance to Ureaplasma spp. is occurring in UP+ women at risk of PTB.

Whole blood flow cytometric analysis of Ureaplasma-stimulated monocytes from pregnant women.

We hypothesised that circulating monocytes of women with vaginal colonisation with Ureaplasma spp., genital microorganisms known to cause inflammation-driven preterm birth, would elicit a tolerised cytokine response to subsequent in vitro Ureaplasma parvum serovar 3 (UpSV3) stimulation. Using multi-parameter flow cytometry, we found no differences with regard to maternal colonisation status in the frequency of TNF-α-, IL-6-, IL-8- and IL-1ß-expressing monocytes in response to subsequent UpSV3 stimulation (P > 0.10 for all cytokines). We conclude that vaginal Ureaplasma spp. colonisation does not specifically tolerise monocytes of pregnant women towards decreased responses to subsequent stimulation.

Superior survival and proliferation after transplantation of myoblasts obtained from adult mice compared with neonatal mice.

BACKGROUND: Myoblast transfer therapy (MTT) is a strategy designed to compensate for the defective gene in myopathies such as Duchenne muscular dystrophy (DMD). Experimental MTT in the mdx mouse (an animal model of DMD) has used donor myoblasts derived from mice of various ages; however, to date, there has been no direct quantitative comparison between the efficacy of MTT using myoblasts isolated from adult and neonate donor muscle. METHODS: Donor normal male myoblasts were injected into Tibialis Anterior muscles of dystrophic female host mice and the survival and proliferation of male myoblasts quantitated using Y-chromosome specific real-time quantitative polymerase chain reaction. The survival of late preplate (PP6) myoblasts derived from neonatal (3-5 days old) or adult (6-8 weeks old) donor mice after MTT were compared. The influence of the number of tissue culture passages, on survival post-MTT, was also evaluated for both types of myoblasts. RESULTS: Surprisingly, superior transplantation efficiency was observed for adult-derived compared with neonatal myoblasts (both early and late passage). Extended expansion (>17 passages) in tissue culture resulted in inferior survival and proliferation of both adult and neonatal myoblasts; however, proliferation of early passage myoblasts (both adult and neonate) was evident between 3 weeks and 3 months. CONCLUSIONS: Myoblasts derived from neonatal mice were inferior for transplantation, and early passage donor myoblasts from adult mice are recommended for MTT in this model.

A comparison between real-time quantitative PCR and DNA hybridization for quantitation of male DNA following myoblast transplantation.

The transplantation of muscle precursor cells (myoblasts) is a potential therapy for Duchenne muscular dystrophy. A commonly used method to detect cell survival is quantitation of the Y chromosome following transplantation of male donor cells into female hosts. This article presents a direct comparison between real-time quantitative PCR (Q-PCR) and the DNA hybridization (slot-blot) technique for quantitation of Y chromosome DNA. Q-PCR has a significantly greater linear quantitation range and is up to 40-fold more sensitive at low concentrations of male DNA, detecting as little as 1 ng of male DNA in each female tibialis anterior (TA) muscle. At high male DNA concentrations, accurate quantitation by Q-PCR is 2.5 times higher than the maximum possible with slot-blot. In conclusion, Q-PCR has a higher dynamic range and is more efficient than slot-blot analysis for the detection of donor cell engraftment in a transsexual transplantation model.

Muscle-derived stem cells: implications for effective myoblast transfer therapy.

Stem cells have been proposed as a wonder solution for tissue repair in many situations and have attracted much attention in the media for both their therapeutic potential and ethical implications. In addition to the excitement generated by embryonic stem cells, research has now identified a number of stem cells within adult tissues which pose much more realistic targets for therapeutic interventions. Myoblast transfer therapy (MTT) has long been viewed as a potential therapy for the debilitating muscle-wasting disorder Duchenne Muscular Dystrophy. This technique relies on the transplantation of committed muscle precursor cells directly into the muscle fibres but has had little success in clinical trials. The recent discovery of a population of cells within adult muscle with stem cell-like characteristics has interesting implications for the future of such putative cell transplantation therapies. This review focuses on the characterization and application of these potential muscle-derived stem cells (MDSC) to MTT.