Tumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action. Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival. When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after ex vivo treatment with TLR-ligation. Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.

Loss of anticancer natural killer (NK) cell function in patients with acute myeloid leukemia (AML) is associated with fatal disease progression and remains poorly understood. Here, we demonstrate that AML blasts isolated from patients rapidly inhibit NK cell function and escape NK cell-mediated killing. Transcriptome analysis of NK cells exposed to AML blasts revealed increased CREM expression and transcriptional activity, indicating enhanced cyclic adenosine monophosphate (cAMP) signaling, confirmed by uniform production of the cAMP-inducing prostanoid prostaglandin E2 (PGE2) by all AML-blast isolates from patients. Phosphoproteome analysis disclosed that PGE2 induced a blockade of lymphocyte-specific protein tyrosine kinase (LCK)–extracellular signal-regulated kinase signaling that is crucial for NK cell activation, indicating a 2-layered escape of AML blasts with low expression of NK cell-activating ligands and inhibition of NK cell signaling. To evaluate the therapeutic potential to target PGE2 inhibition, we combined Fcγ-receptor-mediated activation with the prevention of inhibitory PGE2 signaling. This rescued NK cell function and restored the killing of AML blasts. Thus, we identify the PGE2-LCK signaling axis as the key barrier for NK cell activation in 2-layered immune escape of AML blasts that can be targeted for immune therapy to reconstitute anticancer NK cell immunity in patients with AML.

Breast cancer, one of the most common cancers in women, is classified by the expression of hormone receptors and the growth factor receptor HER2, which is important for personalised tumour treatment with HER2-targeted therapies. Tumour biopsies are required for histopathological diagnosis of HER2 expression by breast cancer cells but are subject to sampling error. In this study, we present a method for identifying and analysing cancer-derived EVs from plasma for the detection of HER2 expression in breast cancer without the need for additional processing steps. We detected nano-sized particles through an optimised flow cytometry approach that allows for the identification of HER2-expressing EVs and quantification of their HER2 expression levels. In a clinical study of 115 breast cancer patients, this optimised flow cytometric analysis detected a range of 1.3 to 50 × 10³ HER2⁺EVs per µl of plasma. The number of HER2⁺EVs did not correlate directly with tumour size, grade, or metastasis. However, computational integration of data from the quantification of HER2^pos EVs per µl/plasma and their HER2 expression levels on a single EV basis allowed for the reliable identification of HER2 expression levels in tumours. Our results reveal the potential for analysing cancer-derived EVs from plasma for the diagnosis and personalised therapy in breast cancer patients.

Extracellular vesicles (EVs) are released from all cells of the body. They are considered to mirror the state of the cells from which they are released and circulate in the blood, suggesting a possible use of EV analysis for diagnostic purposes. Here, we report that the analysis of single EVs by flow cytometry can detect infection of cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying expression of the SARS-CoV-2 spike (S) protein on the surface of EVs and the cellular origin of EVs by detecting cell-type-specific markers such as troponin (cTNT1) for cardiomyocytes. In coronavirus-associated disease 19 (COVID-19) patients, we detected a direct correlation of the frequencies of circulating S-expressing EVs, but not of cTNT/S-co-expressing EVs, with the subsequent development of a severe disease course. Detection of circulating S-expressing EVs indicates widespread SARS-CoV-2 infection in the body, which may contribute to the immune pathogenesis that triggers tissue and organ damage in COVID-19. Our findings suggest that detecting circulating viral antigen-expressing EVs may provide crucial predictive information on infection-associated disease courses in situations of a future viral pandemic. IMPORTANCE: The ability to predict which patients infected with the SARS-CoV-2 virus will develop severe disease remains a significant clinical challenge. The present study demonstrates that EVs in the peripheral blood, carrying the SARS-CoV-2 spike protein, can be detected by flow cytometry and serve as early biomarkers of disease progression. In contradistinction to PCR or serology, this method provides insight into systemic viral spread and potential organ involvement. The early identification of spike-positive EVs at the time of hospital admission has the potential to facilitate the timely identification of high-risk patients, thereby enhancing the efficacy of triage and subsequent care. This approach may also be of value in terms of facilitating a more rapid and precise response to future virus pandemics.