Background Tumor-associated macrophages (TAM) are expanded and exhibit tumor-promoting properties within the tumor microenvironment. Current methods to study TAM have not been replicated across cancer types and often do not include exogenous growth factors from the tumor, a key factor in TAM differentiation in vivo. Methods In this study, an in vitro method to generate monocyte- derived TAM using tumor- conditioned media (TCM) and a cytokine cocktail containing IL-4, IL-10, and M-CSF was utilized to study the phenotype, morphology, and function of TAM across multiple cancer types. TCM was generated from two breast cancer cell lines and an Epstein-Barr virus-positive lymphoma cell line. The properties of in vitro generated TAM were compared to in vitro generated M1 and M2- like macrophages and TAM isolated from patients with cancer. Results TAM generated in this fashion displayed an increase in CD163/CD206 co-expression compared to M2- like macrophages (87 and 36%, respectively). TAM generated in vitro exhibited increased transcript levels of the functional markers IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase compared to in vitro generated M2-like macrophages. Functionally, in vitro generated TAM inhibited the proliferation of T cells (47% decrease from M1-like macrophages) and the production of IFN-γ by natural killer cells was inhibited (44%) when co-cultured with TAM. Furthermore, in vitro generated TAM secreted soluble factors that promote the growth and survival of tumor cells. Conclusions Limited access to patient TAM highlights the need for methods to generate TAM in vitro. Our data confirm that monocyte-derived TAM can be generated reliably using TCM plus the cytokine cocktail of IL-4, IL-10, and M-CSF. Given the ability of TAM to inhibit immune cell function, continued study of methods to deplete or deactivate TAM in the setting of cancer are warranted. Electronic supplementary material The online version of this article (10.1186/s40425-019-0622-0) contains supplementary material, which is available to authorized users.

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An inflammatory microenvironment has been shown to play an important role in the growth and metastasis of tumors. The NLRP3 inflammasome is a multi-protein complex of the innate immune system that is responsible for the production of the potent inflammatory cytokine IL-1β. Tumor-associated macrophages (TAM) are an expanded population of immune cells found in the tumor microenvironment that can promote the initiation and metastasis of tumor cells. Their presence has been correlated with disease burden, highlighting the therapeutic potential of targeting this population. However, to date clinically relevant pharmacologic strategies to target TAM remain elusive. Here, we show that in vitro generated TAM harbor NLRP3 inflammasome components and produce IL-1β. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), is in clinical use for the treatment of B-cell malignancies. We report that BTK is expressed by human in vitro generated TAM and murine macrophages and that it physically associates with the NLRP3 inflammasome. Furthermore, ibrutinib is able to inhibit BTK phosphorylation in TAM generated in vitro. Treatment of TAM with ibrutinib significantly impaired the ability of these cells to produce IL-1β. The present study provides evidence that BTK physically associates with the NLRP3 inflammasome and that inhibition of BTK with ibrutinib can impair the production of IL-1β by in vitro generated TAM. Thus, ibrutinib could potentially be of clinical use in abrogating inflammation-associated cancer progression and the immunesuppressive effects of myeloid cells within the tumor microenvironment.

Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction as inhibited cells had a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora Kinase (A/B) activity and phosphorylation of the downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.

Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) along with the observations of others suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 inhibited cells had a significant increase in unstable kinetochore-microtubule attachments due to impaired error correction of syntelic attachments. We propose that MKLP2 mediates kinetochore microtubule attachment stability by regulating Aurora Kinase and a downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.

Despite innovations in therapy and surgery, glioblastoma remains one of the deadliest tumor types with a 5-year survival rate of approximately 5%. There is a desperate need for new therapeutic strategies to block the growth and survival of glioblastoma. To circumvent toxicities associated with traditional antimitotics, there has been a growing interest in developing therapies that impede mitosis-specific microtubule functions, such as the inhibition of kinesins, mitotic motor proteins that travel along microtubules. Our work with motor kinesin-like protein 2 (MKlp2), in combination with reports from published literature, suggest a previously undescribed role for MKlp2 in early mitosis that would make its inhibition effective at impeding glioblastoma cell growth without neurologic side effects. The objective of this study was to further define this novel function of MKlp2 and to determine the efficacy of MKlp2 inhibition in glioblastoma. Our data indicate that MKlp2 inhibition induced a spindle assembly checkpoint-mediated arrest, suggesting that function of MKlp2 is important for recovery progression to anaphase in addition to its well-known function in late mitosis. In addition, MKlp2 inhibition with a small molecule MKlp2 inhibitor significantly decreased short-term and long-term survival of human glioblastoma patient-derived lines. Furthermore, our data indicate that it is loss of this early mitotic function that is responsible for the anti-proliferative effects caused by MKlp2 inhibition. Ongoing work will continue to delineate the mechanism of MKlp2 in early mitosis and determine the effect of small molecule MKlp2 inhibition in vivo, all the while exploring a new therapeutic strategy for glioblastoma

Myeloid derived suppressor cells (MDSCs) antagonize antitumor immune responses, and limit the efficacy of immune based therapies for cancer. As a result, MDSCs have garnered attention as therapeutic targets. Unfortunately, there has been limited success in translating agents targeting MDSCs to the clinic. Brd4 is an epigenetic reader and is itself a therapeutic target in oncology due to its ability to regulate the expression of oncogenes such as Myc. In addition, Brd4 is known to regulate inflammatory cytokine production and innate immune responses including myeloid cell function. As a result, it was hypothesized that inhibition of Brd4 would impact MDSC function or expansion. In multiple tumor models (EMT6, 4T1, LLC, and C26) Brd4 inhibitors, both experimental (JQ1) and those in clinical development (PLX51107 and PLX2853) significantly reduced the abundance of total, PMN, and M-MDSC subsets within the tumor and spleen as measured by flow cytometry and IHC (p< 0.05). Furthermore, Nanostring gene expression analysis of EMT6 tumors identified a significant reduction in the abundance signature of neutrophils and macrophages with PLX51107 further confirming a reduction of myeloid cells in the presence of a Brd4 inhibitor (p< 0.01). The Nanostring analysis also showed a significant reduction of IL6 mRNA in PLX51107 treated tumors, and this was confirmed at the protein level by ELISA in the serum of EMT6 and 4T1 tumor bearing mice (p< 0.05). However, exogenous IL6 could not rescue MDSC levels in vivo or in vitro suggesting Brd4 inhibition might be directly affecting MDSCs. This was confirmed by showing that PLX51107 significantly inhibited the expansion of MDSCs from mouse bone marrow cells or healthy donor PBMCs cultured in IL6 and GMCSF as measured by flow cytometry (p<0.05). Furthermore, mouse and FACS sorted MDSCs from patients with melanoma or bladder cancer were found to undergo apoptosis when treated with PLX51107 as measured by flow cytometry (annexin and caspase3, p< 0.05), IF, and western blot. In addition, a pancaspase inhibitor rescued both mouse and patient derived MDSCs from apoptosis in the presence of PLX51107. Further investigation with specific caspase inhibitors showed this effect relied mostly on activation of the intrinsic apoptosis pathway and caspase9. The importance of Brd4 to MDSC survival was further confirmed using a LysMCre Brd4 floxed mouse model. Finally, depletion of MDSCs in vivo with PLX51107 significantly enhanced the efficacy of anti-PDL1 therapy as compared to either agent alone (p< 0.05 in EMT6, 4T1, and LLC tumor models). These results identify Brd4 and the TXNIP/ASK1 apoptosis pathway as novel regulators of MDSC survival, and provide evidence to further investigate Brd4 inhibitors in combination with immune based therapies for solid tumors. Citation Format: Andrew Stiff, Himanshu Savardekar, Robert Wesolowski, Megan Duggan, Luke Scarberry, Brooke Benner, Darren Wethington, Gabby Lapurga, Steven Sun, Jack Hedberg, Logan Good, William E. Carson. Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3414.