MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor-immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor-induced gr-MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF-β1-mediated generation of CD4(+)CD25(+)FoxP3(+) iTregs. Furthermore, gr-MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4(+) cells by gr-MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD-1 and PD-L1 signaling, or COX-2. These findings thus indicate that gr-MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.

A high fat diet coincides with elevated levels of bile acids. This elevation of bile acids, particularly deoxycholic acid (DCA), has been strongly associated with the development of colon cancer. Conversely, ursodeoxycholic acid (UDCA) may have chemopreventive properties. Although structurally similar, DCA and UDCA present different biological and pathological effects in colon cancer progression. The differential regulation of cancer by these two bile acids is not yet fully understood. However, one possible explanation for their diverging effects is their ability to differentially regulate signaling pathways involved in the multistep progression of colon cancer, such as the epidermal growth factor receptor (EGFR) mitogen-activated protein kinase (MAPK) pathway. This review will examine the biological effects of DCA and UDCA on colon cancer development, as well as the diverging effects of these bile acids on the oncogenic signaling pathways that play a role in colon cancer development, with a particular emphasis on bile acid regulation of the EGFR-MAPK pathway.

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional antigen presenting cells has been the foundation for the development and utilization of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the non-conventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4+ Th-1, but not Th-2, Th-17 cells or Treg are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs (Th-1 KDCs) was dependent on inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. Importantly, Th-1 KDCs were capable of presenting the acquired antigens from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations open new possibilities for the application of KDCs in cancer immunotherapy.

BackgroundLive-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.ResultsPresented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.ConclusionsThis method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.Electronic supplementary materialThe online version of this article (10.1186/s13008-018-0039-z) contains supplementary material, which is available to authorized users.

Background:The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79A . Association of double-negative B cells with clinical data including gender, age, smoking status, tumor diagnosis and pathologic differentiation status were also examined using the logistic regression analysis for age and student's t-test for all other variables. Associations with other B cell subpopulations were examined using Spearman's rank correlation. Results:We observed that double-negative B cells were frequently abundant in lung tumors compared to normal adjacent controls (13 out of 15 cases), and in some cases made up a substantial proportion of the total B cell compartment. The presence of double-negative cells was also found to be inversely related to the presence of affinitymatured B cells within the tumor, Spearman's coefficient of − 0.76. Conclusions:This study is the first to observe the presence of CD27 − IgD − double-negative B cells in human NSCLC and that this population is inversely correlated with traditional affinity-matured B cell populations.

Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

Overexpression of PD‐L1 (CD274) on tumor cells may represent a hallmark of immune evasion, and overexpression has been documented in several tumors including cutaneous squamous cell carcinoma (cSCC). While PD‐L1/PD‐1 activity in the skin has been primarily described in inflammatory models, our goal was to examine PD‐L1 expression in human keratinocytes exposed to UV irradiation. We assessed PD‐L1 expression in human sun‐protected (SP) and sun‐damaged (SD) skin, actinic keratosis (AK), and cSCC using IHC and protein microarray. Both methods found low baseline levels of PD‐L1 in SP and SD skin and significantly increased expression in cSCC. Next, we examined PD‐L1 expression in acute models of UV exposure. In human SP skin exposed to 2‐3 MED of UV (n = 20), epidermal PD‐L1 was induced in 70% of subjects after 24 h (P = 0.0001). SKH‐1 mice exposed to acute UV also showed significant epidermal PD‐L1 induction at 16, 24 and 48 h. A time‐ and dose‐dependent induction of PD‐L1 was confirmed in cultured human keratinocytes after UV, which was markedly reduced in the presence of MEK/ERK, JNK or STAT3 inhibitors. These findings suggest that UV induces upregulation of PD‐L1 through established, pharmacologically targetable stress‐signaling pathways in keratinocytes.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.