CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma.

Approximately 50% of advanced melanomas harbor activating BRAF V600E mutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (T regs ). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a Braf V600E/+ /Pten -/- graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of T regs and tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.

T Cells and CDDO-Me Attenuate Immunosuppressive Activation of Human Melanoma-Conditioned Macrophages.

Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro tri-culture system of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells, we now show that activated T cells and the synthetic triterpenoid the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) attenuate immune suppression. Surface expression of CD206, CD16 and CD163 on melanoma-conditioned macrophages was inhibited by the addition of T cells, suggesting relief of immuno-suppressive macrophage activation. We also demonstrated that addition of CDDO-Me to tri-cultures enhanced T cell-mediated reductions in CCL2, VEGF and IL-6 production in a contact-independent manner. Because these results suggest CDDO-Me alters melanoma-conditioned macrophage activation, we interrogated CDDO-Me-mediated changes in macrophage signaling pathway activation. Our results indicated that CDDO-Me inhibited phosphorylation of STAT3, a known inducer of TAM activation. Collectively, our studies suggest that activated T cells and CDDO-Me synergistically relieve immune suppression in melanoma cultures and implicate the potential utility of CDDO-Me in the treatment of melanoma.

Chia seed-assisted separation and detection of polyvinyl chloride microplastics in water via gas chromatography mass spectrometry.

Chia seeds were used to significantly improve the separation efficiency of polyvinyl chloride (PVC) microplastics from water samples via centrifugation. Upon hydration, the mucilage of chia seeds were able to capture PVC microplastics with sizes ranging from tens to hundreds of micrometers. Since PVC microplastics contained di-2-etylhexyl phthalate (DEHP) as a plasticizer (verified via Fourier transform infrared spectrometry), DEHP was used as an indicator in the subsequent quantification via gas chromatography - mass spectrometry (GC-MS) analysis. Specifically after verifying the DEHP peak in the GC spectrum using DEHP reference standard as a positive control, the GC spectral area of that peak was used to quantify the amount of DEHP in the sample. Using nominal operation settings at 10 min and 1000 rpm with 100 mg of chia seeds, the separation efficiency could be improved by 5 times (500%) as compared to the absence of chia seeds. Furthermore, chia seeds were also compatible with simulated synthetic wastewater samples. Most importantly, the use of chia seeds did not interfere with GC-MS quantification protocol and accuracy. The result suggested the proposed method can be used as a simple screening tool of microplastics entering wastewater treatment plant, even though a series of follow-up studies are needed in future.

The combined effect of granulocyte-colony stimulating factor (G-CSF) treatment and exercise in rats with spinal cord injury.

Objective: To identify that the combined G-CSF and treadmill exercise is more effective in functional recovery after spinal cord injury (SCI).Design: Rats were divided into 4 groups: a SCI group treated with G-CSF (G-CSF group, n = 6), a SCI group treated with treadmill exercise plus G-CSF (G-CSF/exercise group, n = 6), a SCI group with treadmill exercise (exercise group, n = 6), and a SCI group without treatments (control group, n = 6). We performed laminectomy at the T8-10 spinal levels with compression injury of the spinal cord in all rats. G-CSF (20 µg/ml) was administered intraperitoneally for 5 consecutive days after SCI in G-CSF and G-CSF/exercise groups. From one week after surgery, animals in G-CSF/exercise and exercise groups received 30 min of exercise 5 days per week for 4 weeks. Functional recoveries were assessed using the Basso, Beattie, and Bresnahan (BBB) scale and the inclined plane test. Five weeks after SCI, hematoxylin and eosin staining for cavity size and immunohistochemistry for glial scar formation and neuro-regeneration factor expression were conducted.Setting: Inha University School of medicine, Incheon, KoreaResults: Rats in G-CSF/exercise group showed the most effective functional recovery in the BBB scale and the inclined plane test, and spinal cord cavity size by injury were the smallest, and immunohistochemistry revealed expression of higher BDNF (brain-derived neurotrophic factor) and VEGF (vascular endothelial growth factor) and lower GFAP (glial fibrillary acidic protein) than others.Conclusion: Combined treatment provided more effective neuroplasty and functional recovery than individual treatments.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Removal of copper, nickel and chromium mixtures from metal plating wastewater by adsorption with modified carbon foam.

In this study, the characterizations and adsorption efficiencies for chromium, copper and nickel were evaluated using manufacture-grade Fe2O3-carbon foam. SEM, XRD, XRF and BET analyses were performed to determine the characteristics of the material. Various pore sizes (12-420 µm) and iron contents (3.62%) were found on the surface of the Fe2O3-carbon foam. Fe2O3-carbon foam was found to have excellent adsorption efficiency compared to carbon foam for mixed solutions of cationic and anionic heavy metals. The adsorption capacities for chromium, copper and nickel were 6.7, 3.8 and 6.4 mg/g, respectively, which were obtained using a dual exponential adsorption model. In experiments with varying dosages of the Fe2O3 powder, no notable differences were observed in the removal efficiency. In a fixed-bed column test, Fe2O3-carbon foam achieved adsorption capacities for chromium, copper and nickel of 33.0, 12.0 and 9.5 mg/g, respectively, after 104 h. Based on these results, Fe2O3-carbon foam was observed to be a promising material for treatment of plating wastewater.

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.

Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Expression of heat shock protein 105 and 70 in malignant melanoma and benign melanocytic nevi.

BACKGROUND: Heat shock proteins (HSPs) restore immature proteins or denatured proteins, thus protecting cells. Also, the expression of some HSPs is elevated substantially in malignant tumors, but the expression of HSPs in association with melanoma has yet to be studied. Therefore, we examined the expression patterns of HSP 70 and 105 in melanoma, benign melanocytic nevi and normal human skin. METHODS: Two specimens of malignant melanoma, two of benign melanocytic nevi and six of normal human skin were analyzed using Western blot analysis for expression of HSP 70 and 105. In another set, 16 specimens of malignant melanoma, 24 of benign melanocytic nevi and eight of normal human skin were analyzed for the expression of HSP 105 using immunohistochemical studies. RESULTS: The Western blot analysis showed that HSP 70 was overexpressed in all three types. But, the HSP 105 was hardly expressed in normal human skin and benign melanocytic nevi. However, in malignant melanoma, the HSP 105 was overexpressed, and immunohistochemical examination of HSP 105 showed a result similar to that of Western blot analysis. CONCLUSIONS: In our study, HSP 105 is thought to be a more relevant tumor-associated antigen in malignant melanoma than is HSP 70.