Dataset of cell viability and analytes released by cancer cell lines exposed to low pH and conditioned medium.

Cancer cells influence their microenvironment by secreting factors that promote tumour growth and survival while evading immune-mediated destruction. We previously determined the expression of secreted factors in breast and oesophageal squamous cell carcinoma cell lines (MCF-7 and WHCO6, respectively) using Luminex assays. These cells were subsequently treated with low pH medium to mimic in vivo acid exposure, and the effects on cell viability, proliferation, and secretion of cytokines, chemokines and growth factors were described [1]. Here, we present the datasets from these experiments in addition to data obtained from treating cell lines with conditioned medium from apoptotic cell cultures.

The effect of acute acid exposure on immunomodulatory protein secretion, cell survival, and cell cycle progression in tumour cell lines.

Cancer develops when multiple systems fail to suppress uncontrolled cell proliferation. Breast cancers and oesophageal squamous cell carcinoma (OSCC) are common cancers prone to genetic instability. They typically occur in acidic microenvironments which impacts on cell proliferation, apoptosis, and their influence on surrounding cells to support tumour growth and immune evasion. This study aimed to evaluate the impact of the acidic tumour microenvironment on the production of pro-tumorigenic and immunomodulatory factors in cancer cell lines. Multiple factors that may mediate immune evasion were secreted including IL-6, IL-8, G-CSF, IP-10, GDF-15, Lipocalin-2, sICAM-1, and myoglobin. Others, such as VEGF, FGF, and EGF that are essential for tumour cell survival were also detected. Treatment with moderate acidity did not significantly affect secretion of most proteins, whereas very low pH did. Distinct differences in apoptosis were noted between the cell lines, with WHCO6 being better adapted to survive at moderate acid levels. Conditioned medium from acid-treated cells stimulated increased cell viability and proliferation in WHCO6, but increased cell death in MCF-7. This study highlights the importance of acidic tumour microenvironment in controlling apoptosis, cell proliferation, and immune evasion which may be different at different anatomical sites. Immunomodulatory molecules and growth factors provide therapeutic targets to improve the prognosis of individuals with cancer.

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay.

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.

The acidic tumour microenvironment: Manipulating the immune response to elicit escape.

The success of cancer treatment relies on the composition of the tumour microenvironment which is comprised of tumour cells, blood vessels, stromal cells, immune cells, and extracellular matrix components. Barriers to effective cancer treatment need to be overcome, and the acidic microenvironment of the tumour provides a key target for treatment. This review intends to provide an overview of the effects that low extracellular pH has on components of the tumour microenvironment and how they contribute to immune escape. Further, potential therapeutic targets will be discussed.

Patients Awaiting Renal Transplantation in South Africa: The Need for Highly Sensitive Laboratory Testing and Immunological Risk Stratification.

BACKGROUND: Suitable laboratory testing prior to transplantation ensures that a patient's immunological risk is understood and allows the transplantation team to make the correct decisions on donor-recipient pairing. Preformed donor-specific anti-HLA antibodies mediate rejection in transplanted patients. Improved laboratory testing is needed to increase the chances of successful organ allocation. In this study, we show how Luminex™ single antigen assays are vital in determining immunological risk to transplantation and in predicting waiting time to transplantation. METHODS: Potential recipients awaiting renal transplantation (deceased donor) were tested using Luminex™ single antigen testing. HLA class I and II antibody specificities were determined, and patient demographics were analyzed for each blood group. Antibody reactivity was compared between patients who were transplanted or not transplanted in each blood group. Immunological risk to successful transplantation was determined retrospectively for all patients. RESULTS: Patient demographics were reported for the cohort. More males await renal transplantation than females (male:female ratio of 1.84:1). The median age of the patients was similar between the blood groups (between 41 and 44.5 years), as was the median time spent on the waiting list (between 3 - 4 years). Patients who were transplanted had significantly reduced HLA antibody expression compared to those who were not transplanted (total median reactivity 2 versus 8%, p = 0.0006). Patients were retrospectively stratified according to HLA antibody expression as a surrogate marker for immunological risk. Those in the high-risk group spent longer on the waiting list and had a low chance of being transplanted. CONCLUSIONS: Risk stratification can be used as a tool in predicting likelihood of successful transplantation. Laboratories in South Africa should move towards implementing highly sensitive, highly specific diagnostic assays in order to improve successful organ allocation. This will aid in standardizing testing algorithms in order to transplant more recipients.

Luminex-based virtual crossmatching for renal transplantation in South Africa.

BACKGROUND: Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient's complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively evaluated the use of Luminex anti-human leukocyte antigen (HLA) antibody detection technology, often termed 'virtual crossmatching', compared with the flow cytometric crossmatch, for predicting graft outcome in renal transplant patients. METHODS: Sixty-four recipients were crossmatched against multiple donors during their routine work-up for transplant (111 crossmatches); 17 of these patients received transplants during the study period. Anti-HLA antibody detection was performed using Luminex technology and the results were compared with the flow cytometric crossmatch results and with short-term graft success. RESULTS: Compared with flow cytometric crossmatch results, the sensitivity and specificity of Luminex virtual crossmatching was 85.7% and 90.7% for the T-cell crossmatch and 100% and 87.2% for the B-cell crossmatch. Both the sensitivity and specificity of Luminex for predicting short-term graft success were 100%. CONCLUSIONS: Strong evidence is provided that single-antigen assays provide improved sensitivity to detect clinically relevant anti-HLA antibodies and can reliably be used to predict short-term graft success. We recommend incorporation of single-antigen Luminex methodology into the routine work-up algorithm of renal transplant recipients in South Africa.

Multi-analyte profiling of ten cytokines in South African HIV-infected patients with Immune Reconstitution Inflammatory Syndrome (IRIS).

BACKGROUND: Immune reconstitution inflammatory syndrome (IRIS) is an important complication of HAART in sub-Saharan Africa, where opportunistic infections (OIs) including mycobacteria and cryptococcus are common. The immune system's role in HIV infected patients is complex with cytokine expression strongly influencing HIV infection and replication. METHODS: We determined the expression patterns of 10 cytokines by Luminex multi-analyte profiling in 17 IRIS nested case-control pairs participating in a prospective South African cohort initiating anti-retroviral therapy. RESULTS: Interferon-gamma (IFN-γ) expression was significantly elevated in IRIS cases compared to controls (median 9.88 pg/ml versus 2.68 pg/ml, respectively, P = 0.0057), while other cytokines displayed non-significant differences in expression. Significant correlation was observed between IL-6, IL-10, and IFN-γ expression in the IRIS patients. CONCLUSIONS: Significantly increased expression levels of IFN-γ suggest that this cytokine possibly plays a role in IRIS pathology and is a potential diagnostic marker.