Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation.

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1ß and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.

Circulating low density neutrophils of breast cancer patients are associated with their worse prognosis due to the impairment of T cell responses.

Neutrophils are prominent immune components of tumors, having either anti-tumor (N1) or pro-tumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN are rare in non-pathological conditions, but frequent in cancer, exhibiting a pro-tumor phenotype. These findings have been mainly demonstrated in animal models, thus proper validation in humans is still imperative. Here, we observed that LDN were increased in the blood of breast cancer (BC) patients, particularly with metastatic disease. Within the population of non-metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than patients with a good response. The higher incidence of LDN in BC patients with severe disease or resistance to treatment can be explained by their pro-tumor/immunosuppressive characteristics. Moreover, the percentage of LDN in BC patients' blood was negatively correlated with activated cytotoxic T lymphocytes and positively correlated with immunosuppressive regulatory T cells. The ability of LDN to spoil anti-tumor immune responses was further demonstrated ex vivo. Hence, this study reveals the potential of LDN as a biomarker of BC response to treatment and opens new avenues for developing new immunotherapies.

Expression of HLA-DR in Cytotoxic T Lymphocytes: A Validated Predictive Biomarker and a Potential Therapeutic Strategy in Breast Cancer.

Neoadjuvant chemotherapy (NACT) is common in breast cancer (BC) treatment, though more than half of the patients lack an effective response. Therefore, new predictive biomarkers and alternative therapies are crucial. Previously, we proposed HLA-DR-expressing cytotoxic T lymphocytes (CTLs) as a potential biomarker of the response to NACT. To validate this observation and further investigate these cells, 202 BC patients were enrolled. Flow cytometry analyses were performed in 61 biopsies and 41 blood samples pre-NACT and 100 non-NACT tumor samples. All the patients were followed up for 34 months. Blood-isolated immune cells were cultured with BC cell lines in a 3D system. We confirmed that HLA-DR level in CTLs is a highly sensitive, specific, and independent biomarker to predict response to NACT and developed a predictive probability model. This biomarker was also associated with progression-free survival, regardless of the treatment. The clinical observations are substantiated by the anti-tumor properties of HLA-DR-expressing CTLs. Intriguingly, HLA-DR level in CTLs can be modulated ex vivo, boosting their capacity to kill tumor cells synergistically with doxorubicin. Thus, HLA-DR expression in CTLs is a validated tool to select patients that will actually benefit from NACT, and its stimulation might be a novel therapeutic approach for BC.

Establishment of a 3D Co-culture With MDA-MB-231 Breast Cancer Cell Line and Patient-Derived Immune Cells for Application in the Development of Immunotherapies.

3D cell culture including different cell types, such as immune cells, is a representative platform that mimics the tumor microenvironment. Here we disclose an easy-to-handle 3D co-culture protocol using a scaffold-free technique with the breast cancer cell line MDA-MB-231 and breast cancer patient-derived immune cells from peripheral blood. The method presented is simple, less time-consuming and less expensive when compared to other 3D techniques. Additionally, this is an optimized protocol for the establishment of a 3D system for this cell line, which is normally seen as challenging to spontaneously form spheroids. The addition of patient-derived immune cells to the cancer cells' spheroid allows the study of the crosstalk between both cell types, as well as the assessment of individual therapeutic approaches to intensify the antitumor immune response. In fact, with this model, we observed that patients' immune cells exhibit a wide range of antitumor responses and we further demonstrated that it is possible to manipulate the less effective ones with a canonical stimulus, as a proof-of-concept, in order to improve their ability to lower the viability of tumor cells. Therefore, this platform could be applied for a personalized immune-based drug screening, with results after a maximum of 10 days of culture, in order to develop more tailored breast cancer treatments and ameliorate patients' survival rate.

HLA-DR in Cytotoxic T Lymphocytes Predicts Breast Cancer Patients' Response to Neoadjuvant Chemotherapy.

Prediction of breast cancer response to Neoadjuvant Chemotherapy (NACT) is an urgent need to promptly direct non-responder patients to alternative therapies. Infiltrating T lymphocytes, namely cytotoxic T lymphocytes (CTLs) have been appointed as predictors of response. However, cancer cells have the ability to dampen CTLs' activity and thus, the prognostic value of the CTLs, per se, is debatable. Here, we disclose that more than the occurrence of CTLs, it is their activation state, revealed by HLA-DR expression, that can accurately predict response to NACT. Flow cytometry analysis of breast cancer biopsies showed that the frequency of CTLs and other lymphocytes were similar regardless disease stage and between NACT responders and non-responders. However, only breast cancer patients without axillary lymph node metastasis and NACT responders have HLA-DRhi CTLs. Interestingly, HLA-DR levels in tumor CTLs is correlated with HLA-DR levels in systemic CTLs. These HLA-DR+ CTLs produce IFN-γ and Granzyme B, enlightening their effector and probable anti-tumor activity profile. Moreover, the level of HLA-DR in CTLs is negatively correlated with the level of HLA-DR in T regulatory lymphocytes and with immunosuppressive and pro-tumor molecules in the tumor microenvironment. Hence, HLA-DR levels in CTLs is a highly sensitive and specific potential predictive factor of NACT-response, which can be assessed in blood to guide therapeutic decisions.

Population genetics-informed meta-analysis in seven genes associated with risk to dengue fever disease.

Population genetics theory predicted that rare frequent markers would be the main contributors for heritability of complex diseases, but meta-analyses of genome-wide association studies are revealing otherwise common markers, present in all population groups, as the identified candidate genes. In this work, we applied a population-genetics informed meta-analysis to 10 markers located in seven genes said to be associated with dengue fever disease. Seven markers (in PLCE1, CD32, CD209, OAS1 and OAS3 genes) have high-frequency and the other three (in MICB and TNFA genes) have intermediate frequency. Most of these markers have high discriminatory power between population groups, but their frequencies follow the rules of genetic drift, and seem to have not been under strong selective pressure. There was a good agreement in directional consistency across trans-ethnic association signals, in East Asian and Latin American cohorts, with heterogeneity generated by randomness between studies and especially by low sample sizes. This led to confirm the following significant associations: with DF, odds ratio of 0.67 for TNFA-rs1800629-A; with DHF, 0.82 for CD32-rs1801274-G; with DSS, 0.55 for OAS3-rs2285933-G, 0.80 for PLCE1-rs2274223-G and 1.32 for MICB-rs3132468-C. The overall genetic risks confirmed sub-Saharan African populations and descendants as the best protected against the severer forms of the disease, while Southeast and Northeast Asians are the least protected ones. European and close neighbours are the best protected against dengue fever, while, again, Southeast and Northeast Asians are the least protected ones. These risk scores provide important predictive information for the largely naïve European and North American regions, as well as for Africa where misdiagnosis with other hemorrhagic diseases is of concern.

How many diseases is triple negative breast cancer: the protagonism of the immune microenvironment.

Triple negative breast cancer (TNBC) is a type of breast cancer (BC) that does not express the oestrogen and the progesterone receptors and the human epidermal growth factor receptor type 2 (HER2). Since there are no positive markers to reliably classify TNBC, these tumours are not yet treated with targeted therapies. Perhaps for this reason they are the most aggressive form of breast carcinomas. However, the clinical observation that these patients do not carry a uniformly dismal prognosis, coupled with data coming from pathology and epidemiology, suggests that this negative definition is not capturing a single clinical entity, but several. We critically evaluate this evidence in this paper, reviewing clinical and epidemiological data and new studies that aim to subclassify TNBC. Moreover, evidence on the role of tumour infiltrating lymphocytes (TILs) on TNBC progression, response to chemotherapy and patient outcome have been published. The heterogeneity, observed even at TILs level, highlights the idea that TNBC is much more than a single disease with a unique treatment. The exploration of the immune environment present at the tumour site could indeed help in answering the question 'How many diseases is TNBC' and will help to define prognosis and eventually develop new therapies, by stimulating the immune effector cells or by inhibiting immunological repressor molecules. In this review, we focus on the prospect of the patient's diverse immune signatures within the tumour as potential biomarkers and how they could be modulated to fight the disease.