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Correlation of Hsp70 Serum Levels with Gross Tumor Volume and Composition of Lymphocyte Subpopulations in Patients with Squamous Cell and Adeno Non-Small Cell Lung Cancer.

Gunther S, Ostheimer C, Stangl S, Specht HM, Mozes P, Jesinghaus M, Vordermark D, Combs SE, Peltz F, Jung MP, Multhoff G - Front Immunol (2015)

Bottom Line: Heat-shock protein 70 (Hsp70) is frequently found on the plasma membrane of a large number of malignant tumors including non-small cell lung cancer (NSCLC) and gets released into the blood circulation in lipid vesicles.A significant correlation of serum Hsp70 levels with the gross tumor volume was shown for adeno and squamous cell NSCLC.However, significantly elevated ratios of activated CD69(+)/CD94(+) NK cells that are associated with low serum Hsp70 levels were observed only in patients with squamous cell lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München (TUM) , München , Germany.

ABSTRACT
Heat-shock protein 70 (Hsp70) is frequently found on the plasma membrane of a large number of malignant tumors including non-small cell lung cancer (NSCLC) and gets released into the blood circulation in lipid vesicles. On the one hand, a membrane (m)Hsp70-positive phenotype correlates with a high aggressiveness of the tumor; on the other hand, mHsp70 serves as a target for natural killer (NK) cells that had been pre-stimulated with Hsp70-peptide TKD plus low-dose interleukin-2 (TKD/IL-2). Following activation, NK cells show an up-regulated expression of activatory C-type lectin receptors, such as CD94/NKG2C, NKG2D, and natural cytotoxicity receptors (NCRs; NKp44, NKp46, and NKp30) and thereby gain the capacity to kill mHsp70-positive tumor cells. With respect to these results, the efficacy of ex vivo TKD/IL-2 stimulated, autologous NK cells is currently tested in a proof-of-concept phase II clinical trial in patients with squamous cell NSCLC after radiochemotherapy (RCT) at the TUM. Inclusion criteria are histological proven, non-resectable NSCLC in stage IIIA/IIIB, clinical responses to RCT and a mHsp70-positive tumor phenotype. The mHsp70 status is determined in the serum of patients using the lipHsp70 ELISA test, which enables the quantification of liposomal and free Hsp70. Squamous cell and adeno NSCLC patients had significantly higher serum Hsp70 levels than healthy controls. A significant correlation of serum Hsp70 levels with the gross tumor volume was shown for adeno and squamous cell NSCLC. However, significantly elevated ratios of activated CD69(+)/CD94(+) NK cells that are associated with low serum Hsp70 levels were observed only in patients with squamous cell lung cancer. These data might provide a first hint that squamous cell NSCLC is more immunogenic than adeno NSCLC.

No MeSH data available.


Related in: MedlinePlus

Relative amounts of lymphocytes (%) in healthy human individuals and patients with squamous cell and adeno NSCLC. Comparison of the percentage of peripheral blood lymphocytes (PBL) in healthy human individuals (n = 10) and patients with squamous cell (n = 25) and adeno (n = 18) NSCLC at diagnosis (patient collective #1); **p < 0.01, ***p < 0.001 (Mann–Whitney U-test). Graph below: gate R1 refers to the population of PBL which is analyzed by FACS, G2 refers to the population of granulocytes.
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Figure 3: Relative amounts of lymphocytes (%) in healthy human individuals and patients with squamous cell and adeno NSCLC. Comparison of the percentage of peripheral blood lymphocytes (PBL) in healthy human individuals (n = 10) and patients with squamous cell (n = 25) and adeno (n = 18) NSCLC at diagnosis (patient collective #1); **p < 0.01, ***p < 0.001 (Mann–Whitney U-test). Graph below: gate R1 refers to the population of PBL which is analyzed by FACS, G2 refers to the population of granulocytes.

Mentions: In order to determine the proportion of different lymphocyte subpopulations, flow cytometric (FACS) analysis was performed using freshly collected EDTA blood (1.4 ml). Therefore, blood (100 μl) was transferred into 14 test tubes and then fluorescently labeled antibodies were added. The antibody combinations that were used for the FACS analysis are summarized in Table 3. After an incubation time of 15 min in the dark, the tubes were centrifuged for 5 min at 500 g at room temperature after adding 2 ml of PBS/10% FCS washing buffer. In order to eliminate erythrocytes, cells were incubated with lysing buffer (1:9 dilution of BD Lysing Solution Cat. 3490202 with millipore H2O) for 10 min at the room temperature in the dark. The respective percentages of B, T, and NK cell subpopulations are defined as the proportion of cells within the lymphocyte gate R1 (see Figure 3). For the determination of regulatory T cells, buffer A (1:10 dilution of component A with H2O) was added to the respective tubes. After two washing steps, cells were permeabilized with buffer C (1:50 dilution of buffer A with component B) for 30 min in the dark. Following another two washing steps, a PE-conjugated antibody directed against the intracellular transcription factor forkhead box P3 (FoxP3) was added for another 30 min. After another two washing steps, 5 × 104 cells were analyzed on a FACScalibur instrument (Becton Dickinson, Heidelberg, Germany).


Correlation of Hsp70 Serum Levels with Gross Tumor Volume and Composition of Lymphocyte Subpopulations in Patients with Squamous Cell and Adeno Non-Small Cell Lung Cancer.

Gunther S, Ostheimer C, Stangl S, Specht HM, Mozes P, Jesinghaus M, Vordermark D, Combs SE, Peltz F, Jung MP, Multhoff G - Front Immunol (2015)

Relative amounts of lymphocytes (%) in healthy human individuals and patients with squamous cell and adeno NSCLC. Comparison of the percentage of peripheral blood lymphocytes (PBL) in healthy human individuals (n = 10) and patients with squamous cell (n = 25) and adeno (n = 18) NSCLC at diagnosis (patient collective #1); **p < 0.01, ***p < 0.001 (Mann–Whitney U-test). Graph below: gate R1 refers to the population of PBL which is analyzed by FACS, G2 refers to the population of granulocytes.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4629690&req=5

Figure 3: Relative amounts of lymphocytes (%) in healthy human individuals and patients with squamous cell and adeno NSCLC. Comparison of the percentage of peripheral blood lymphocytes (PBL) in healthy human individuals (n = 10) and patients with squamous cell (n = 25) and adeno (n = 18) NSCLC at diagnosis (patient collective #1); **p < 0.01, ***p < 0.001 (Mann–Whitney U-test). Graph below: gate R1 refers to the population of PBL which is analyzed by FACS, G2 refers to the population of granulocytes.
Mentions: In order to determine the proportion of different lymphocyte subpopulations, flow cytometric (FACS) analysis was performed using freshly collected EDTA blood (1.4 ml). Therefore, blood (100 μl) was transferred into 14 test tubes and then fluorescently labeled antibodies were added. The antibody combinations that were used for the FACS analysis are summarized in Table 3. After an incubation time of 15 min in the dark, the tubes were centrifuged for 5 min at 500 g at room temperature after adding 2 ml of PBS/10% FCS washing buffer. In order to eliminate erythrocytes, cells were incubated with lysing buffer (1:9 dilution of BD Lysing Solution Cat. 3490202 with millipore H2O) for 10 min at the room temperature in the dark. The respective percentages of B, T, and NK cell subpopulations are defined as the proportion of cells within the lymphocyte gate R1 (see Figure 3). For the determination of regulatory T cells, buffer A (1:10 dilution of component A with H2O) was added to the respective tubes. After two washing steps, cells were permeabilized with buffer C (1:50 dilution of buffer A with component B) for 30 min in the dark. Following another two washing steps, a PE-conjugated antibody directed against the intracellular transcription factor forkhead box P3 (FoxP3) was added for another 30 min. After another two washing steps, 5 × 104 cells were analyzed on a FACScalibur instrument (Becton Dickinson, Heidelberg, Germany).

Bottom Line: Heat-shock protein 70 (Hsp70) is frequently found on the plasma membrane of a large number of malignant tumors including non-small cell lung cancer (NSCLC) and gets released into the blood circulation in lipid vesicles.A significant correlation of serum Hsp70 levels with the gross tumor volume was shown for adeno and squamous cell NSCLC.However, significantly elevated ratios of activated CD69(+)/CD94(+) NK cells that are associated with low serum Hsp70 levels were observed only in patients with squamous cell lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München (TUM) , München , Germany.

ABSTRACT
Heat-shock protein 70 (Hsp70) is frequently found on the plasma membrane of a large number of malignant tumors including non-small cell lung cancer (NSCLC) and gets released into the blood circulation in lipid vesicles. On the one hand, a membrane (m)Hsp70-positive phenotype correlates with a high aggressiveness of the tumor; on the other hand, mHsp70 serves as a target for natural killer (NK) cells that had been pre-stimulated with Hsp70-peptide TKD plus low-dose interleukin-2 (TKD/IL-2). Following activation, NK cells show an up-regulated expression of activatory C-type lectin receptors, such as CD94/NKG2C, NKG2D, and natural cytotoxicity receptors (NCRs; NKp44, NKp46, and NKp30) and thereby gain the capacity to kill mHsp70-positive tumor cells. With respect to these results, the efficacy of ex vivo TKD/IL-2 stimulated, autologous NK cells is currently tested in a proof-of-concept phase II clinical trial in patients with squamous cell NSCLC after radiochemotherapy (RCT) at the TUM. Inclusion criteria are histological proven, non-resectable NSCLC in stage IIIA/IIIB, clinical responses to RCT and a mHsp70-positive tumor phenotype. The mHsp70 status is determined in the serum of patients using the lipHsp70 ELISA test, which enables the quantification of liposomal and free Hsp70. Squamous cell and adeno NSCLC patients had significantly higher serum Hsp70 levels than healthy controls. A significant correlation of serum Hsp70 levels with the gross tumor volume was shown for adeno and squamous cell NSCLC. However, significantly elevated ratios of activated CD69(+)/CD94(+) NK cells that are associated with low serum Hsp70 levels were observed only in patients with squamous cell lung cancer. These data might provide a first hint that squamous cell NSCLC is more immunogenic than adeno NSCLC.

No MeSH data available.


Related in: MedlinePlus