Mitochondrial Oxygenation During Cardiopulmonary Bypass: A Pilot Study

ObjectiveAdequate oxygenation is essential for the preservation of organ function during cardiac surgery and cardiopulmonary bypass (CPB). Both hypoxia and hyperoxia result in undesired outcomes, and a narrow window for optimal oxygenation exists. Current perioperative monitoring techniques are not always sufficient to monitor adequate oxygenation. The non-invasive COMET® monitor could be a tool to monitor oxygenation by measuring the cutaneous mitochondrial oxygen tension (mitoPO2). This pilot study examines the feasibility of cutaneous mitoPO2 measurements during cardiothoracic procedures. Cutaneous mitoPO2 will be compared to tissue oxygenation (StO2) as measured by near-infrared spectroscopy.Design and MethodThis single-center observational study examined 41 cardiac surgery patients requiring CPB. Preoperatively, patients received a 5-aminolevulinic acid plaster on the upper arm to enable mitoPO2 measurements. After induction of anesthesia, both cutaneous mitoPO2 and StO2 were measured throughout the procedure. The patients were observed until discharge for the development of acute kidney insufficiency (AKI).ResultsCutaneous mitoPO2 was successfully measured in all patients and was 63.5 [40.0–74.8] mmHg at the surgery start and decreased significantly (p < 0.01) to 36.4 [18.4–56.0] mmHg by the end of the CPB run. StO2 at the surgery start was 80.5 [76.8–84.3]% and did not change significantly. Cross-clamping of the aorta and the switch to non-pulsatile flow resulted in a median cutaneous mitoPO2 decrease of 7 mmHg (p < 0.01). The cessation of the aortic cross-clamping period resulted in an increase of 4 mmHg (p < 0.01). Totally, four patients developed AKI and had a lower preoperative eGFR of 52 vs. 81 ml/min in the non-AKI group. The AKI group spent 32% of the operation time with a cutaneous mitoPO2 value under 20 mmHg as compared to 8% in the non-AKI group.ConclusionThis pilot study illustrated the feasibility of measuring cutaneous mitoPO2 using the COMET® monitor during cardiothoracic procedures. Moreover, in contrast to StO2, mitoPO2 decreased significantly with the increasing CPB run time. Cutaneous mitoPO2 also significantly decreased during the aortic cross-clamping period and increased upon the release of the clamp, but StO2 did not. This emphasized the sensitivity of cutaneous mitoPO2 to detect circulatory and microvascular changes

RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers

Ubiquitin ligase STUB1 destabilizes IFNγ-receptor complex to suppress tumor IFNγ signaling

The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identify STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This is corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response is increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells, but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome

A method to assess the clinical significance of unclassified variants in the BRCA1 and BRCA2 genes based on cancer family history

Introduction Unclassified variants (UVs) in the BRCA1/BRCA2 genes are a frequent problem in counseling breast cancer and/or ovarian cancer families. Information about cancer family history is usually available, but has rarely been used to evaluate UVs. The aim of the present study was to identify which is the best combination of clinical parameters that can predict whether a UV is deleterious, to be used for the classification of UVs. Methods We developed logistic regression models with the best combination of clinical features that distinguished a positive control of BRCA pathogenic variants (115 families) from a negative control population of BRCA variants initially classified as UVs and later considered neutral (38 families). Results The models included a combination of BRCAPRO scores, Myriad scores, number of ovarian cancers in the family, the age at diagnosis, and the number of persons with ovarian tumors and/ or breast tumors. The areas under the receiver operating characteristic curves were respectively 0.935 and 0.836 for the BRCA1 and BRCA2 models. For each model, the minimum receiver operating characteristic distance (respectively 90% and 78% specificity for BRCA1 and BRCA2) was chosen as the cutoff value to predict which UVs are deleterious from a study population of 12 UVs, present in 59 Dutch families. The p. S1655F, p. R1699W, and p. R1699Q variants in BRCA1 and the p. Y2660D, p. R2784Q, and p. R3052W variants in BRCA2 are classified as deleterious according to our models. The predictions of the p. L246V variant in BRCA1 and of the p. Y42C, p. E462G, p. R2888C, and p. R3052Q variants in BRCA2 are in agreement with published information of them being neutral. The p. R2784W variant in BRCA2 remains uncertain. Conclusions The present study shows that these developed models are useful to classify UVs in clinical genetic practic

Methylcholanthrene-Induced Sarcomas Develop Independently from NOX2-Derived ROS

<div><p>Reactive oxygen species (ROS) produced by the inducible NADPH oxidase type 2 (NOX2) complex are essential for clearing certain infectious organisms but may also have a role in regulating inflammation and immune response. For example, ROS is involved in myeloid derived suppressor cell (MDSC)- and regulatory T cell (T_reg) mediated T- and NK-cell suppression. However, abundant ROS produced within the tumor microenvironment, or by the tumor itself may also yield oxidative stress, which can blunt anti-tumor immune responses as well as eventually leading to tumor toxicity. In this study we aimed to decipher the role of NOX2-derived ROS in a chemically (by methylcholanthrene (MCA)) induced sarcoma model. Superoxide production by NOX2 requires the p47^phox (NCF1) subunit to organize the formation of the NOX2 complex on the cell membrane. Homozygous mutant mice (NCF1*^/*) have a functional loss of their super oxide burst while heterozygous mice (NCF1*^/+) retain this key function. Mice harboring either a homo- or a heterozygous mutation were injected intramuscularly with MCA to induce sarcoma formation. We found that NOX2 functionality does not determine tumor incidence in the tested MCA model. Comprehensive immune monitoring in tumor bearing mice showed that infiltrating immune cells experienced an increase in their oxidative state regardless of the NOX2 functionality. While MCA-induced sarcomas where characterized by a T_reg and MDSC accumulation, no significant differences could be found between NCF1*^/* and NCF1*^/+ mice. Furthermore, infiltrating T cells showed an increase in effector-memory cell phenotype markers in both NCF1*^/* and NCF1*^/+ mice. Tumors established from both NCF1*^/* and NCF1*^/+ mice were tested for their <i>in vitro</i> proliferative capacity as well as their resistance to cisplatin and radiation therapy, with no differences being recorded. Overall our findings indicate that NOX2 activity does not play a key role in tumor development or immune cell infiltration in the chemically induced MCA sarcoma model.</p></div

Growth of MCA induced tumors is not affected by NCF1 mutation.

<p>(A) NCF1*^/* (n = 6) and NCF1*^/+ (n = 10) mice were tested for their ability to generate superoxide through stimulation of NOX2 with PMA. (B) NCF1*^/* and NCF1*^/+ mice (n = 54, 27 per group) were injected with 125 μg MCA dissolved in 25 μl of corn oil. Tumor incidence was followed through palpation of the injected muscle site. (C) Tumors were resected from MCA injected NCF1*^/* (n = 6) and NCF1*^/+ (n = 6) mice and passaged once in WT C57Bl/6 mice prior to transplantation of 5³ mm tumor into four WT C57Bl/6 (n = 48) mice in which (Ci) survival (Cii) and tumor growth were monitored.</p

T cells are not affected differently in NCF1*^/* compared to NCF1*^/+ tumor bearing mice with regard to their CD4/CD8 ratio, activation status or memory phenotype.

<p>(A) T cells were gated as singlet cells that were not stained for dead cell marker but positive for CD3. (B) The ratio of CD4+ to CD8+ was gated in blood, spleen and tumor in NCF1*^/* and NCF1*^/+ mice, with the dotted line representing the ratio found in WT C57Bl/6 spleenocytes. (C) Activation marker CD69 was stained on blood, spleen and tumor resident T cells. (D) Blood, spleen and tumor resident T cells were stained for CD62L as well as CD44 to indicate their memory phenotype.</p

Non-functional NOX2 does not change susceptibility to cisplatin or radiotherapy.

<p>(Ai) Cells were seeded in E-plates and treated with 5μg/ml of cisplatin at log phase growth. (Aii) Rate of cell death after addition of cisplatin was compared between NCF1 tumor cell lines, derived from MCA induced tumor in NCF1*^/* and NCF1*^/+ mice. (B) NCF1 tumor cell lines were treated with 10 gray and morphology of the cells was followed for three days. (Ci) 100 tumor cells were seeded in plates and treated with 2 gray and followed over 6 or 9 days. Metabolic activity was measured with XTT as an indication of survival with treatment over control (Cii) indicating survival.</p

Oxidative state does not differ in immune cell subsets or tumors from NCF1*^/* and NCF1*^/+ tumor bearing mice.

<p>(A) Cells were gated on live followed by CD45 positive both CD3 negative followed by CD11b positive and GR1 positive for MDSC, CD45 positive CD3 positive indicated T cells and CD45 negative live cells were considered tumor cells. (B) DHR-123 MFI of MDSCs as well (C) T cells and (D) tumor cells were determined in NCF1*^/* (n = 4) and NCF1*^/+ (n = 5) tumor bearing mice.</p