An investigation into the immunomodulatory activities of human placental protein 14 (PP14).

PP14 has been shown to suppress the incorporation of [3]H-Thymidine into both mitogenically and allogeneically stimulated lymphocytes in a dose dependent manner. The suppressive activity was shown to be specific, in that PP14 did not affect cellular viability, nor interact with the mitogen phytohaemagglutinin (PHA). Flow cytometric analysis indicated that PP14 had no effect on the expression of the Tac antigen, the transferrin receptor or HLA-DR molecules on the surface of stimulated lymphocytes. Neither did PP14 affect the interaction of interleukin-2 (IL-2) with its cell surface receptor. The suppressive activity was partially reversed by the addition of exogenous IL-2. PP14 inhibited the production of IL-2 from mitogenically stimulated lymphocytes and led to a small, but significant reduction in soluble IL-2 receptor release. Radiolabel binding studies and IL-2 dose response curves indicated that PP14 affected the affinity of the IL-2 receptor on PHA stimulated lymphocytes. This was supported by the observation that PP14 increased the level of cell surface-associated IL-2 on stimulated lymphocytes. There was a small inhibition of gamma interferon levels early in the culture period. PP14 had no effect on the CD4/CD8 ratio following stimulation and was not found to be associated with the cell surface, nor mask cell surface expression of the CD2 antigen.These data suggest that the immunosuppressive activity of PP14 may, in part, be mediated via a modulation of the functional, high affinity IL-2 receptor. It is not known as yet whether such an activity is effective at the level of induction of the receptors or whether the primary control is at another level of the response. PP14 may have implications in the study of implantation and fertility and prove of wider interest in the field of transplantation biology and the control of the immune response

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner

Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research

SPAG5 as a prognostic biomarker and chemotherapy sensitivity predictor in breast cancer: a retrospective, integrated genomic, transcriptomic, and protein analysis

Background: Although the use of proliferation markers/profiles has been recommended when choosing the appropriate systemic-treatment for breast cancer (BC), the best molecular-marker/test that should be used needs to be identified. Methods: To identify factors that drive proliferation and its associated features in BC an artificial neural network (ANN) based integrative data-mining methodology was applied to three cohorts [(Nottingham-discovery (ND), Uppsala and METABRIC (Molecular Taxonomy of Breast Cancer International Consortium)]. The most prominent genes in the resulting interactome-map were then identified. Given that SPAG5 was associated with many features of proliferation, featured prominently in the interactome-map and has a fundamental role in mitotic-progression,, we hypothesized that it could be a better indicator of proliferation activity. (BC). Subsequently to test if it could provide a more accurate guide for the delivery of systemic therapies in BC, we investigated the clinico-pathological utility of SPAG5: gene copy number aberrations (CNAs); mRNA and protein expression, in over 10,000 BCs. Integrated analysis of SPAG5-gene CNAs, transcript and protein expression was conducted in the ND cohort (n=171) and validated in the METABRIC cohort (n=1980). In addition, the associations of SPAG5 CNAs, transcript and/or protein with BC specific survival (BCSS), disease free survival (DFS) and/or distant relapse free survival (DRFS) were analysed in multiple cohorts including Uppsala (n=249), METABRIC, three-untreated lymph node (LN) negative cohorts (n=684), a combined multicentre clinical data set (n=5439), Nottingham historical early-stage-primary BC (Nottingham-HES-BC; n=1650), Nottingham oestrogen receptor (ER) negative BC (n=697), Nottingham anthracycline-Neoadjuvant-chemotherapy (Nottingham-AC-Neo-ACT; n=200), and MD Anderson Cancer Centre Taxane/anthracycline (MDACC-T/AC-Neo-ACT; n=508) cohorts. The association of SPAG5 transcript and protein expression with pathological response rate (pCR) were also tested in [MDACC-T/AC-Neo-ACT (n=508) and the phase II trial NCT00455533; n=253)] and [Nottingham-AC-Neo-ACT (n=200)] cohorts; respectively. Findings: SPAG5 gene gain/amplification at the Ch17q11·2 locus was found in 10.4% of BC (206/1980 (; METABRIC) and was reported in 19·4% of PAM50-HER2 (46/237) and 17·8% of PAM50-LumB (87/488). SPAG5-CNA gain/amplification and high SPAG5-transcript and SPAG5-protein were associated with increased risk of death from BC [Uppsala; (HR (CI 95%): 1·50 (1·18-1·92); p=0·00010, METABRIC; (HR (CI 95%): 1·68 (1·40-2·01) p<0·0001), and Nottingham-HSE-BC; (HR (CI 95%): 1·68 (1·32-2·12), p<0·0001); respectively]. Multivariable Cox regression models, including other validated-prognostic factors, (Uppsala: age, size, LN-stage, genomic grade index (GGI), ER, TP53 mutation and MKi67; METABRIC: age, size, LN-stage, histologic-grade, ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), hormone-therapy, chemotherapy, interaction term of SPAG5 and both chemotherapyy and hormonotherapy; Desmedt-untreated LN- cohort: ER, Nottingham prognostic index (NPI), 76-gene prognostic signature (Veridex) and Adjuvant-Online (AOL); Nottingham-HES-BC: menopausal status, size, LN- stage, histologic-grade, ER, PR, HER2, ki67, hormone-therapy, chemotherapy, interaction term of SPAG5 and both chemotherapy[y and hormonotherapy), showed that high SPAG5-transcript and high SPAG5-protein were associated with shorter BCSS [Uppsala: (HR (CI 95%): 1·62 (1·03-2·53) p=0·036); METABRIC: (HR (CI 95%): 1·27 (1·02-1·58) p=0·034); Desmedt-untreated LN- cohort: (HR (CI 95%): 2·34 (1·24-4·42) p=0·0090), and Nottingham-HES-BC (HR (CI 95%): 1·73 (1·23-2·46) p=0·0020); respectively]. In ER-negative-BC with high SPAG5-protein, administration of anthracycline-adjuvant-chemotherapy had reduced the risk of death by 60% compared to chemotherapy-naive (HR (95% CI): 0·37 (0·20-0·60); p=0·0010). A multivariable Cox regression analysis, which included other validated prognostic factors for chemotherapy (e.g., menopausal status, size, lymph node stage, histologic grade, ER, PR, HER2, Bcl2, chemotherapy, interaction term of SPAG5 and both chemotherapy[y), revealed that SPAG5-transcript+ was independently associated with decreased risk of DRFS after receiving Taxane/anthracycline-Neo-ACT [MDACC-T/AC-Neo-ACT: (HR (CI 95%): 0·68 (0·48-0·97); p=0·0070)]. In multivariable logistic regression analysis, both SPAG5-transcript+ and SPAG5-protein+ and were independent predictors for higher pCR after combination-cytotoxic chemotherapy [MDACC-T/AC-Neo-ACT: (OR (95% CI) 1·71 (1·07-2·74); p=0·024), and Nottingham-AC-Neo-AC: (OR (95% CI): 8·75 (2·42-31); p=0·0010); respectively]. Interpretation: SPAG5 is a novel amplified gene on Ch17q11.2 in PAM50-LumB and PAM-HER2 BC, and its transcript and protein products are independent prognostic and predictive biomarkers, with potential clinical utility as a biomarker for combination cytotoxic chemotherapy sensitivity, especially in ER- BC

Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells

Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/2≥7/3<5/5<4/6<3/7<2/8). In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC50 of Hsp70=85 μg/ml) and hypoxic (EC50 of Hsp70=55 μg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 μg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.—Schilling, D., Gehrmann, M., Steinem, C., De Maio, A., Pockley, A. G., Abend, M., Molls, M., Multhoff, G. Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells

Kinetics of TPP internalization.

<p>(Left panel) Flow cytometric analysis of memHsp70 positive human breast cancer lines MCF7, MDA-MB-231, T47D reveals an accumulation of the fluorescence intensity after incubation with CF-labeled TPP at 37°C between 1 and 60 min (solid lines). At 4°C (dashed lines) mean fluorescence intensity remained low within the same time frame. Cell lines with a high percentage of Hsp70 membrane positive cells (MCF7, MDA-MB-231) exhibit a higher and more rapid uptake of TPP, whereas the cell line with low Hsp70 membrane expression (T47D) exhibits only a low uptake of TPP. (Right panel) Confocal microscopy images were analyzed to provide a total count of fluorescent spots per cell after incubation with the TPP at 37°C for 30 min. Although fluorescent spots progressively accumulated in all three cell lines, this was most apparent in the MCF7 and MDA-MB-231 cell lines that express higher levels of memHsp70 than T47D cells.</p

TPP co-localizes with mitochondria after 90 min.

<p>MCF7, MDA-MB-231, and T47D cells were incubated with CF-labeled TPP (green) for 90 min, stained with the mitochondrial detection dye Mito-ID (purple) and then imaged using confocal microscopy. A proportion of internalized TPP co-localizes with mitochondria in all three tumor cell lines (Pearson’s coefficient: MCF7 r = 0.855, MDA-MB-231 r = 0.585, T47D r = 0.813). The Manders’ M1 coefficient was used to estimate the proportion of total TPP that co-localizes with mitochondria (40% in MCF7 cells, M1 = 0.407; 44% in MDA-MB-231 cells, M1 = 0.443). In contrast, 14% of TPP was co-localized to mitochondria in T47D cells (M1 = 0.141). These findings correlate with the differential Hsp70 membrane expression levels of the respective cancer cell lines. Images are representative single frames. Objective 63×; scale bar 10 µm.</p

Tumor cell lines expressing memHsp70 are able to bind TPP at 4°C.

<p>The memHsp70 status of a panel of human breast, colon, melanoma, lung, head & neck tumor cell lines was assessed using the cmHsp70.1 antibody (top row histograms). Incubation of tumor cells with carboxyfluorescein (CF)-labeled TPP at 4°C (bottom row histograms) results in a similar binding profile to that of cmHsp70.1 antibody in all tumor cell lines; grey, isotype controls, open histograms, Hsp70 specific reagents. The numbers in the histograms show the proportion of Hsp70 membrane-positively stained cells.</p