Cognate Nonlytic Interactions between CD8+ T Cells and Breast Cancer Cells Induce Cancer Stem Cell-like Properties.

Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8+ T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8+ T cells also increased the proportion of stem cell-like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell-like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8+ T cells enables increased tumor growth and metastasis in BALB/cnu/nu mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. SIGNIFICANCE: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.

Evaluation of Sentinel Lymph Node Biopsy and Axillary Lymph Node Dissection for Breast Cancer Treatment Concepts - a Retrospective Study of 1,214 Breast Cancer Patients.

BACKGROUND: Most breast cancer patients require lumpectomy with axillary sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND). The ACOSOG Z0011-trial failed to detect significant effects of ALND on disease-free and overall survival among patients with limited sentinel lymph node (SLN) metastases. Intense dose-dense chemotherapy and supraclavicular fossa radiation (SFR) are indicated for patients with extensive axillary metastases. In this multicentered study, we investigated the relevance of ALND after positive SLNB to determine adequate adjuvant therapy. METHODS: We retrospectively analyzed data from 1,214 patients with clinically nodal negative T1-T2 invasive breast cancer undergoing surgery at Hanau City Hospital Breast cancer center. RESULTS: 681 patients underwent ALND after SLNB. 20 patients (8.5%) from the group with 1 or 2 SLN metastases (n = 236) showed more than 3 lymph node metastases after ALND. 13 patients (31.7%) from the group with more than 2 SLN metastases (n = 41) were diagnosed with a minimum of 4 axillary lymph node metastases after ALND. CONCLUSIONS: In 8.5% of the patients with 1 or 2 SLN metastases, ALND detected more than 3 macrometastases, setting the indication for intense dose-dense chemotherapy and SFR. More than 2 SLN metastases, T stage and grading predict lymph node metastases.

Adenosine-generating ovarian cancer cells attract myeloid cells which differentiate into adenosine-generating tumor associated macrophages - a self-amplifying, CD39- and CD73-dependent mechanism for tumor immune escape.

BACKGROUND: Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo. METHODS: Monocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized "TAM-like" macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4(+) T cells and specific inhibitors. RESULTS: When CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4(+) T cell proliferation which could be rescued via blockade of CD39 or CD73. CONCLUSION: Adenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer.

CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) Cells: Rare Populations in the Human Decidua with Characteristics of MDSC.

PROBLEM: Human pregnancy needs a remarkable local immune tolerance toward the conceptus. Myeloid-derived suppressor cells (MDSC) are important players promoting cancer initiation and progression by suppressing T-cell functions and thus inducing immune tolerance. Therefore, MDSC were expected within decidua. METHODS: Subpopulations of CD33(+) immune cells were isolated from human early pregnancy decidua and characterized phenotypically and functionally by microscopy, FACS analysis, RT-PCR, Western blotting and in the coculture with T cells. RESULTS: Decidua harbors CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) cells which both express arginase, iNOS and IDO and a typical cytokine profile. Both subtypes potently suppress T-cell proliferation and therefore fulfill the criteria of MDSC. CONCLUSION: We characterized a new population of CD33(+) /HLA-DR(neg) and CD33(+) /HLA-DR(+/-) cells in human early pregnancy decidua with properties of classical MDSC and thus potentially being an important player in immune tolerance in pregnancy.

Effects of lobaplatin as a single agent and in combination with TRAIL on the growth of triple-negative p53-mutated breast cancers in vitro.

Lobaplatin as a single agent and in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is investigated in in-vitro models of p53-negative triple-negative breast cancers (TNBCs) and compared with a model of oestrogen receptor-positive p53-positive breast cancer. In addition, the induction of programmed cell death by lobaplatin is further explored. By using cell viability assays and western blotting, the cytotoxic effects of lobaplatin alone and in combination with TRAIL are compared with cisplatin in HCC 1806, HCC 1937, and MCF 7 cells. The multicaspase inhibitor z-VAD-fmk and necrostatin, an inhibitor of necroptosis, are used to demonstrate the mechanism of cell death caused by lobaplatin. Lobaplatin displayed antitumour activity in all three cell lines, which increased time dependently. Cotreatment of lobaplatin and TRAIL induced an increase in cytotoxicity by 30-50% in the different cell lines. The pan-caspase inhibitor z-VAD-fmk as well as necrostatin could weaken but not abolish the cytotoxic effect of lobaplatin and cisplatin. Lobaplatin showed substantial cytotoxic effects in two in-vitro models of p53-mutated TNBC. Cotreatment with TRAIL and platinum agents resulted in increased antitumour activity in the TNBC cell lines investigated. Cell death subsequent to treatment with cisplatin and lobaplatin occurred because of apoptosis. However, caspase-independent mechanisms of programmed cell death were also involved. It was also demonstrated that platinum compounds could induce necroptosis, although to a minor extent.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity.

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

A cell-based luciferase-dependent assay for the quantitative determination of free extracellular adenosine with paracrine signaling activity.

Extracellular adenosine exerts powerful paracrine effects on immune cells. Thus, adenosine signaling has to be strictly regulated. This is achieved by its rapid internalization or enzymatic degradation. Consequently, free adenosine is extremely difficult to measure in cell culture systems and may escape from detection by time-consuming endpoint measurements like high-performance liquid chromatography (HPLC). Therefore, we have now developed a highly sensitive assay which enables the quantification of biologically relevant extracellular adenosine via the activation of an ectopically expressed Adenosine 2a-receptor (ADORA2A) in HEK-293 reporter cells. Binding of the short-lived nucleoside to this receptor induces a cAMP-dependent signal which can be detected via a cAMP-responsive luciferase construct. Tests with exogenously added adenosine confirmed that the resulting luminescence signals correlate with the respective adenosine levels and thus allow quantitative measurements in a range from 20 nM to 80 µM free extracellular adenosine. Inhibition of adenosine uptake by dipyridamole further increased the sensitivity of the assay. We further validated our approach by quantifying the adenosine levels that are generated by regulatory T cells via ectonucleotidase-mediated cleavage of ATP. As expected, values returned to baseline when ADORA2A was inhibited. This confirmed that this new cell-based reporter assay constitutes a biologically relevant, technically easy, versatile, scalable and cost-effective approach that allows the non-radioactive quantification of adenosine as a signaling intermediate.

Reduced cortisol levels in cerebrospinal fluid and differential distribution of 11beta-hydroxysteroid dehydrogenases in multiple sclerosis: implications for lesion pathogenesis.

Relapses during multiple sclerosis (MS) are treated by administration of exogenous corticosteroids. However, little is known about the bioavailability of endogenous steroids in the central nervous system (CNS) of MS patients. We thus determined cortisol and dehydroepiandrosterone (DHEA) levels in serum and cerebrospinal fluid (CSF) samples from 34 MS patients, 28 patients with non-inflammatory neurological diseases (NIND) and 16 patients with other inflammatory neurological diseases (OIND). This revealed that MS patients - in sharp contrast to patients with OIND - show normal cortisol concentrations in serum and lowered cortisol levels in the CSF during acute relapses. This local cortisol deficit may relate to poor local activation of cortisone via 11beta-hydroxysteroid dehydrogenase type 1 (11bHSD1) or to inactivation via 11bHSD2. Accordingly, 11bHSD2 was found to be expressed within active plaques, whereas 11bHSD1 was predominantly detected in surrounding "foamy" macrophages. Our study thus provides new insights into the impaired endogenous CNS cortisol regulation in MS patients and its possible relation to MS lesion pathogenesis. Moreover, an observed upregulation of 11bHSD1 in myelin-loaded macrophages in vitro suggests an intriguing hypothesis for the self-limiting nature of MS lesion development. Finally, our findings provide an attractive explanation for the effectivity of high- vs. low-dose exogenous corticosteroids in the therapy of acute relapses.

Macrophage migration inhibitory factor expression in cervical cancer.

PURPOSE: The glycoprotein macrophage migration inhibitory factor (MIF) is a cytokine that has been shown to promote tumor progression and tumor immune escape in ovarian cancer. The present study investigates MIF in uterine cervical cancer. METHODS: Eighty surgical biopsies (32 cervical dysplasias, 23 in situ carcinomas and 25 invasive carcinomas) of uterine cervical tissue were evaluated immunohistochemically for MIF expression. In uterine cervical cancer cell lines SiHa and CaSki and their respective supernatants, MIF protein expression was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis shows that MIF is clearly overexpressed on the protein level in invasive cervical cancer compared to cervical dysplasias. MIF overexpression was confirmed by RT-PCR in surgical biopsies of invasive cervical cancer. Western blotting reveals that the MIF protein is overexpressed in SiHA und CaSki cervical cancer cell lines, whereas the ELISA reveals that cervical cancer cells secrete MIF. CONCLUSIONS: MIF has been shown to promote tumor immune escape mechanisms in other cancer entities, which makes it an interesting target for cancer therapy, given the known significance of immune mechanisms for uterine cervical cancer. The overexpression of MIF on the protein and mRNA level, as well as its secretion by cervical cancer cells points to a critical role of the protein for the pathogenesis of uterine cervical cancer.