Vita Pašukonienė

Accumulation and biological effects of cobalt ferrite nanoparticles in human pancreatic and ovarian cancer cells

BACKGROUND AND OBJECTIVE Superparamagnetic iron oxide nanoparticles (SPIONs) emerge as a promising tool for early cancer diagnostics and targeted therapy. However, both toxicity and biological activity of SPIONs should be evaluated in detail. The aim of this study was to synthesize superparamagnetic cobalt ferrite nanoparticles (Co-SPIONs), and to investigate their uptake, toxicity and effects on cancer stem-like properties in human pancreatic cancer cell line MiaPaCa2 and human ovarian cancer cell line A2780. MATERIALS AND METHODS Co-SPIONs were produced by Massarts co-precipitation method. The cells were treated with Co-SPIONs at three different concentrations (0.095, 0.48, and 0.95μg/mL) for 24 and 48h. Cell viability and proliferation were analyzed after treatment. The stem-like properties of cells were assessed by investigating the cell clonogenicity and expression of cancer stem cell-associated markers, including CD24/ESA in A2780 cell line and CD44/ALDH1 in MiaPaCa2 cell line. Magnetically activated cell sorting was used for the separation of magnetically labeled and unlabeled cells. RESULTS Both cancer cell lines accumulated Co-SPIONs, however differences in response to nanoparticles were observed between MiaPaCa2 and A2780 cell. In particular, A2780 cells were more sensitive to exposition to Co-SPIONs than MiaPaCa2 cells, indicating that a safe concentration of nanoparticles must be estimated individually for a particular cell type. Higher doses of Co-SPIONs decreased both the clonogenicity and ESA marker expression in A2780 cells. CONCLUSIONS Co-SPIONs are not cytotoxic to cancer cells, at least when used at a concentration of up to 0.95μg/mL. Co-SPIONs have a dose-dependent effect on the clonogenic potential and ESA marker expression in A2780 cells. Magnetic detection of low concentrations of Co-SPIONS in cancer cells is a promising tool for further applications of these nanoparticles in cancer diagnosis and treatment; however, extensive research in this field is needed.

CD8highCD57+ T-cell population as an independent predictor of response to chemoradiation therapy in extensive-stage small cell lung cancer

OBJECTIVES Tangible clinical benefit is achieved in only a relatively small proportion of extensive-stage small cell lung cancer (SCLC) patients receiving current treatment strategies. Therefore, a more personalized use of current and novel treatment approaches is of critical importance. Individualized therapy relies on the identification of specific biomarkers predictive of response to a particular type of cancer treatment. Immune-related parameters emerge as powerful biomarkers among a variety of predictors of clinical response to various types of cancer treatment. PATIENTS AND METHODS Using multicolor flow cytometry, we evaluated a predictive value of CD8(high)CD57(+) T-cell population and its immunosuppressive (FOXP3(+), NKG2A(+)) and cytotoxic (Perforin(+)) subsets in the peripheral blood of extensive-stage SCLC patients (n=82) treated with either chemotherapy-alone (n=24), or chemoradiation therapy (n=42), or receiving best supportive care (n=16). RESULTS The low level (<20%) of CD8(high)CD57(+) T cells within the peripheral blood CD8(+) T-cell population and the low level (<3%) of the immunosuppressive FOXP3-positive subset within the CD8(high)CD57(+) T-cell population were independent predictors of a better response to treatment with chemoradiation therapy, but not with chemotherapy alone or best supportive care. Importantly there was no significant survival difference between SCLC patients who were: (i) treated with chemoradiation, but had an unfavourable immune profile (≥20% of CD8(high)CD57(+) T cells and ≥3% of its FOXP3-positive subset), (ii) treated with chemotherapy alone, or (iii) received best supportive care. CONCLUSIONS We show that only a combination of chemotherapy with radiation therapy offered a considerable survival benefit that was confined to a subset of extensive-stage SCLC patients with a favourable predictive immune profile in the peripheral blood.

Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation

BACKGROUND AND OBJECTIVE The aim of this study was to evaluate the efficiency of proton beam irradiation in pancreatic cancer cell line MIA PaCa-2 and its role in the cell cycle, apoptosis, and formation of histone γH2AX in different reparation times (72-h follow-up). MATERIAL AND METHODS The MIA PaCa-2 pancreatic carcinoma cell line was irradiated with 1.6-Gy proton beam. After irradiation, cell viability was measured colorimetrically, and the cell cycle, apoptosis, and γH2AX expression were evaluated on a FACScan cytometer. RESULTS Low-dose proton beam irradiation had an effect on the MIA PaCa-2 tumor cell line already 1h after exposure, but maximal lethality was reached after 72h postirradiation with a cell viability rate of 24%. The cell cycle went into partial G1/0 arrest, and was released after 72h. The expression of γH2AX was strong and its levels were significantly elevated as late as 48h post radiation. The apoptosis levels increased with post radiation incubation time to reach 79% after 72h. CONCLUSIONS Our data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase.

Post-operative unadjuvanted therapeutic xenovaccination with chicken whole embryo vaccine suppresses distant micrometastases and prolongs survival in a murine Lewis lung carcinoma model

Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patients immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a Bacillus subtilis protein fraction as an adjuvant. Median and overall survival, size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared with untreated control mice. Following primary tumour removal, a course of three subcutaneous vaccinations with xenogeneic chicken embryo vaccine led to significant increase in overall survival rate (100% after 70 days of follow-up vs. 40% in untreated control mice), significant increase in circulating CD8a+ T cells (18.18 vs. 12.6% in untreated control mice), and a significant decrease in the area and incidence of metastasis foci. The xenogeneic rat brain tissue-based vaccine did not improve any of the investigated parameters, despite promising reports in other models. We hypothesize that the proper selection of antigen source (tissue) can constitute an effective immunotherapeutic product.

Quantitative changes within the peripheral blood CD8highCD57+ T-cell subpopulation of patients with advanced renal cell carcinoma

Background. Antitumor immunotherapy strategies, such as cytokine- based or cell-based immunotherapies, are designed to activate immune response against cancer cells, but considering that malignancy may be associated with the expansion of immunosuppressive components of antitumor immunity, it is likely that in such cases activation of the immune system would further enhance activity of these components, leading to more severe suppression of antitumor immune response thus making more favourable conditions for tumor progression. Materials and methods. We studied the expression of biomarkers, representing immunosuppressive (FOXP3) and cytotoxic (perforin, IFN-γ) CD8 high CD57 + T-cell subpopulation functions in the peripheral blood of 34 patients with advanced clear cell renal cell carcinoma (RCC) and 26 controls by multicolour flow cytometry. Results. CD8 high CD57 + T cell subpopulation of all CD8 + T cells in RCC patients was significantly higher compared to age-matched healthy controls (p = 0.003). It was found that the mean percentage of suppressive CD8 high CD57 + FOXP3 + T-cell subset and cytotoxic CD8 high CD57 + Perforin + T-cell subset in the CD8 high CD57 + T-cell subpopulation was significantly increased in RCC patients compared to controls (p = 0.0004 and p = 0.008, respectively). There was no strong and biologically relevant negative correlation between the expression of FOXP3 and Perforin in the peripheral blood CD8 high CD57 + T-cell subpopulation of RCC patients. Conclusions. Subsets of immunosuppressive FOXP3+ T cell and tumor-attacking (cytotoxic) Perforin+ T cells in the CD8 high CD57 + T-cell subpopulation are significantly increased in RCC patients compared to controls. These quantitative rearrangements are independent and individual for each RCC patient.

Quantitative changes of functionally different CD8 h CD57 + T-cell subsets in the peripheral blood of advanced renal cell carcinoma or high-risk melanoma patients@@@Skirtingomis funkcijomis pasižyminčių CD8 h CD57 + T limfocitų subpopuliacijų kiekybiniai pokyčiai išplitusia inkstų ląstelių karcinoma ar didelės rizikos melanoma sergančių pacientų periferiniame kraujyje

Correspondence to: Marius Strioga, Laboratory of Immunology, Institute of Oncology, Vilnius University, P. Baublio 3B, LT-08660, Vilnius, Lithuania. E-mail: marius.strioga@vuoi.lt Introduction. CD8hCD57+ T-cell subpopulation and its functionally diff erent subsets play an important role in antitumour immunity. Th e relation of competitive subsets may infl uence the overall eff ect of CD8hCD57+ T-cell mediated antitumour immunity and determine an individual response to antitumour immunotherapy. Th e aim of this study was to evaluate the proportion of cytotoxic, immunomodulating and immunosuppressive subsets of the CD8hCD57+ T-cell subpopulation in the peripheral blood of cancer patients and agematched healthy controls. Materials and methods. We studied the expression of biomarkers representing the cytotoxic (perforin), immunosuppressive (FOXP3, NKG2A) and immunomodulating (IFNγ) properties of CD8+CD57+ T cells in the peripheral blood of 49 cancer patients: 30 with clear cell renal cell carcinoma (age median 58, range 43–81), 19 with high risk cutaneous melanoma (age median 68, range 45–86) and 26 controls (age median 55, range 41–81) by multicolour fl ow cytometry. Results. Th e percentage of immunosuppressive CD8hCD57+FOXP3+ T-cell subset in CD8hCD57+ T-cell population varied. It was absent in 65% of controls, while only 23% and 26% of such patients were observed in renal cell carcinoma (RCC) and melanoma groups, respectively. Even 40% of RCC and 37% of melanoma patients had a high percentage of CD8hCD57+FOXP3+ T-cell subset, while in the control group we found no such subjects. Th e cytotoxic CD8hCD57+Perforin+ T-cell subset was signifi cantly increased in RCC patients, but showed no relevant rise in melanoma patients, whereas the immunomodulating CD8hCD57+IFNγ+ subset was signifi cantly increased in melanoma patients but showed no relevant rise in RCC patients when compared to controls. Conclusions. Th e amount of various functionally diff erent subsets in CD8hCD57+ T-cell subpopulation varies greatly among cancer patients. Th ese diff erences may infl uence the overall CD8hCD57+ T-cell mediated antitumour immune response and determine an individual response to antitumour immunotherapy.