Critical evaluation of the interaction of reactive oxygen and nitrogen species with blood to inform the clinical translation of nonthermal plasma therapy

Non-thermal plasma (NTP), an ionized gas generated at ambient pressure and temperature, has been an emerging technology for medical applications. Through controlled delivery of reactive oxygen and nitrogen species (ROS/RNS), NTP can elicit hormetic cellular responses, thus stimulating broad therapeutic effects. To enable clinical translation of the promising preclinical research into NTP therapy, a deeper understanding of NTP interactions with clinical substrates is profoundly needed. Since NTP-generated ROS/RNS will inevitably interact with blood in several clinical contexts, understanding their stability in this system is crucial. In this study, two medically relevant NTP delivery modalities were used to assess the stability of NTP-generated ROS/RNS in three aqueous solutions with increasing organic complexities: phosphate-buffered saline (PBS), blood plasma (BP), and processed whole blood. NTP-generated RNS collectively (NO2−, ONOO−), H2O2, and ONOO− exclusively were analyzed over time. We demonstrated that NTP-generated RNS and H2O2 were stable in PBS but scavenged by different components of the blood. While RNS remained stable in BP after initial scavenging effects, it was completely reduced in processed whole blood. On the other hand, H2O2 was completely scavenged in both liquids over time. Our previously developed luminescent probe europium(III) was used for precision measurement of ONOO− concentration. NTP-generated ONOO− was detected in all three liquids for up to at least 30 seconds, thus highlighting its therapeutic potential. Based on our results, we discussed the necessary considerations to choose the most optimal NTP modality for delivery of ROS/RNS to and via blood in the clinical context

Comparisons between the hematocytological counts at different sampling sites.

<p>Hematocytological counts are shown for all comparisons with P<.10 for both the raw and normalized cell counts. The univariate T/W method shows the P value for the two-sided paired t-test ‘T’ (‘Ln’ when a natural logarithm was applied) or Wilcoxon paired-matched signed rank (‘W’) in case normality didn’t hold. The Bonferroni and Benjamini columns show the adjusted significance levels according to the method applied. Sensitivity for outliers was investigated for the raw cell counts. See Materials & Methods for more information.</p><p>Results annotated with ‘*’ are considered significant for the method applied. <i>Sites</i> comparison between sampling sites; <i>ARD</i> average relative difference; <i>PLT</i> platelets; <i>MON</i> monocytes; <i>BAS</i> basophils; <i>NGC</i> neutrophils; <i>LEU</i> leukocytes; <i>RBC</i> red blood cells; <i>HCT</i> hematocrit; <i>RD</i> radial artery vs. dorsal hand veins; <i>RE</i> radial artery vs. antecubital veins; <i>DE</i> dorsal hand veins vs. antecubital veins; <i>NA</i> not applicable.</p>a<p>After omission of outliers the ARD for RE reduced to 3.4% and for DE to 3.7% with significances similar to before.</p

Comparisons between the lymphocyte subpopulation counts at different sampling sites.

<p>Lymphocyte subpopulation counts are shown for all comparisons with P<.10 for both the relative and absolute cell counts. The univariate T/W method shows the P value for the two-sided paired t-test ‘T’ (‘Ln’ when a natural logarithm was applied) or Wilcoxon paired-matched signed rank (‘W’) in case normality didn’t hold. The Bonferroni and Benjamini columns show the adjusted significance levels according to the method applied. Sensitivity for outliers was performed for the relative cell counts. The mixed column shows all comparisons with P<.10 assessed as contrasts within a multivariate analysis for the relative counts for the lymphocyte subpopulations using SAS 9.2. The mixed joint column shows the multivariate P value. See Materials & Methods for more information.</p><p>Results annotated with ‘*’ are considered significant for the method applied. <i>Sites</i> comparison between sampling sites; <i>ARD</i> average relative difference; <i>PLT</i> platelets; <i>NGC</i> neutrophils; <i>WBC</i> white blood cells; <i>RBC</i> red blood cells; <i>HCT</i> hematocrit; <i>RD</i> radial artery vs. dorsal hand veins; <i>RE</i> radial artery vs. antecubital veins; <i>DE</i> dorsal hand veins vs. antecubital veins.</p>a<p>After omission of outliers the P value from the paired t-test was 0.093 with ARD 6.7%. The multivariate analysis showed a P value of 0.014.</p>b<p>After omission of outliers the P value from the paired t-test was 0.11 with ARD 3.3%. The multivariate analysis showed a P value of 0.042.</p

Comparisons between the lymphocyte subpopulation median fluorescence intensities of the skin homing marker CLA at different sampling sites.

<p>Lymphocyte subpopulations with comparisons between sampling sites for the CLA median fluorescence intensities with P<.10 are shown for the raw data and in addition for the data after omission of outliers. Comparisons with P>.10 after omission of outliers and with P<.10 for the raw data are also shown. The univariate T/W method shows the P value for the two-sided paired t-test ‘T’ (‘Ln’ when a natural logarithm was applied) or Wilcoxon paired-matched signed rank (‘W’) in case normality didn’t hold. The Bonferroni and Benjamini columns show the adjusted significance levels according to the method applied See Materials & Methods for more information.</p><p>Results annotated with ‘*’ are considered significant for the method applied. <i>Sites</i> comparison between sampling sites; <i>ARD</i> average relative difference; <i>RD</i> radial artery vs. dorsal hand veins; <i>RE</i> radial artery vs. antecubital veins; <i>DE</i> dorsal hand veins vs. antecubital veins; <i>NO</i> no outliers.</p

Image_2_The prognostic impact of the immune signature in head and neck squamous cell carcinoma.tif

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.</p

Image_1_The prognostic impact of the immune signature in head and neck squamous cell carcinoma.tif

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.</p

Image_3_The prognostic impact of the immune signature in head and neck squamous cell carcinoma.tif

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.</p