Transforming tumor cells into professional antigen-presenting cells using poly(β-amino ester) nanoparticles to deliver CD80-encoding mRNA.

Expression of the Stem Cell Markers Nestin and CD133 on Circulating Melanoma Cells

Targeting Hypoxia-inducible Factor-1α With Tf–PEI–shRNA Complex via Transferrin Receptor–mediated Endocytosis Inhibits Melanoma Growth

Approximately 50% of patients with uveal melanomas develop metastases. Thus, it is important to improve our understanding of how melanoma metastases develop. As part of a uveal melanoma micrometastasis study, we compared the detection rates of immunomagnetically selected (IMS) tumour cells in bone marrow (BM) with positively stained tumour cells using immunocytochemistry (ICC). Bone marrow mononuclear cells were isolated. Immunocytochemistry cytospin preparations were immunocytochemically stained in parallel with two different melanoma antibodies, 9.2.27 and HMB45. Using IMS, melanoma cells were selected from BM mononuclear cell fractions using immunomagnetic beads coated with the 9.2.27 antibody and identified by light microscopy. In cytospin preparations from 226 patients, melanoma cells were detected in 24 (10.6%), 10 with 9.2.27 and 17 with the HMB45 antibody. In three cases, we found positive cells with both antibodies. Six of the 226 (2.6%) patients that stained positively with ICC died with metastatic disease, all also positive with IMS. Sixty-six (29.2%) patients had positive BM samples with IMS at the first examination. Immunomagnetic selection (IMS) was positive in 36.8% of the 57 patients who later developed clinical metastases. Twenty-one IMS-positive patients and 31 IMS-negative patients died of metastases, in total 52 of 226 patients (23.0%). The mortality rate of melanoma metastasis was 24% (6/24) after at least 4 ½ years in ICC-positive patients compared to 38.5% (20/52) in IMS-positive patients. The presence of melanoma cells in BM of patients with uveal melanoma is documented in our study with IMS and ICC. Immunomagnetically selected is more sensitive than ICC in detecting tumour cells in BM. However, statistically, we did not find any prognostic impact of the presence of melanoma cells in BM.

Experiments were performed to compare the ability of ocular and skin melanoma cells to stimulate T cells. Primary melanoma cell lines were obtained from a series of patients with either eye or skin melanoma. The ability of tumor cells to stimulate T cells in the absence of exogenous growth factors was assessed in mixed-lymphocyte tumor cell cultures in which allogeneic lymphocytes were stimulated with irradiated ocular or skin melanoma cells. Expression of HLA class I and class II on tumor cells, in the presence or absence of IFN-gamma, was determined by flow cytometry. The ability of tumor cells to inhibit T-cell proliferation was determined by adding various concentrations of irradiated tumor cells to standard mixed-lymphocyte cultures. Our results indicate that primary skin melanoma cells induce vigorous proliferation of allo-antigen-specific T cells. By contrast, ocular melanoma cells failed to induce significant T-cell proliferation. The failure of ocular melanoma cells to stimulate lymphocyte proliferation was not due to low levels of either class I or class II on tumor cells since tumor cells treated with IFN-gamma expressed high levels of class I and class II but still failed to induce lymphocyte proliferation. Ocular melanoma cells inhibited lymphocyte proliferation, as shown by experiments in which a small number of tumor cells prevented proliferation of T cells in mixed-lymphocyte cultures. Inhibition of lymphocyte proliferation required cell-to-cell contact, and supernatants from tumor cell cultures did not prevent lymphocyte proliferation. Moreover, the ability of ocular melanoma cells to inhibit T-cell proliferation was lost when tumor cells migrated from the eye and formed hepatic metastases. We conclude that there is a fundamental difference in the immunogenicity of ocular and skin melanoma cells. Ocular melanomas, but not primary skin melanomas, are poorly immunogenic tumors that inhibit T-cell proliferation. Our results imply that the immunogenicity of melanoma cells is altered when they develop within the unique ocular micro-environment.

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma.

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.

Topical Application of Cream-emulsified CD86 siRNA Ameliorates Allergic Skin Disease by Targeting Cutaneous Dendritic Cells

2520 Background: Despite the transformational impact of immune checkpoint blockade, many cancer patients do not experience long-term survival. T cells with innate immune signatures can secrete inflammatory cytokines/chemokines and deliver potent cytotoxic signals potentially ideal for tumor immunity. The novel double-stranded RNA sensor RIG-I agonist SLR14 improved the control of murine melanoma. We tested the hypothesis that SLR14 transforms T cells to a cytotoxic state in immunologically “cold” human tumor specimens. Methods: We developed an approach, called PERCEPT, to directly test the response of patient tumor and immune samples to novel and established therapies ex vivo using perturbational single-cell RNA sequencing (Table). We obtained 9 surgical resections from primary or metastatic melanoma and Merkel Cell Carcinoma (MCC) tumors and lymph node metastases and made suspension replicates of tumor and infiltrating immune cell co-cultures. We stimulated for 42-48 hours (Table). We Flourescently Activated Cell Sorted live cells and then barcoded for multiplexed single-cell sequencing using 10x scRNAseq. We used CINEMA-OT to identify factors associated with response and resistance to the perturbations tested. We developed and validated CRISPR-KO MCC and small cell lung cancer (SCLC) cell lines, and co-cultured with CD14+ monocytes or monocyte-derived DCs. Results: Stimulation with RIG-I agonist SLR14 induced expression beyond canonical IFN-stimulated genes in tumor cells, NK cells, and T cells. SLR14 stimulates tumor-infiltrating T cells into antiviral states in tumor-immune co-cultures and primes in vitro T-cell production of IFNγ. However, MCC immune infiltrate responsiveness to IFN or SLR14 was notably decreased compared to the melanoma samples, and perturbational computational analyses with CINEMA-OT identified the cytokine midkine (MDK) associated with nonresponse in MCC. Knockout of MDK restored response to IFN and SLR14 by MCC and SCLC tumor cell lines, as well as co-cultured CD14+ monocytes or monocyte-derived DCs. Conclusions: Our approach revealed that midkine, a multifunctional cytokine, suppresses innate immune sensing of IFN and SLR14 in both tumor and immune cells, disrupting the tumor immunity cycle at multiple points. We show that this effect, while comparatively infrequent in melanoma, is pronounced in MCC and SCLC. Our study thus uses a direct assessment of patient tumor and immune samples to identify a novel resistance mechanism enriched in neuroendocrine tumors MCC and SCLC. Therapeutic class Stimulation Target Clinical development Immune checkpoint inhibitor αPD-1 PD-1 Standard of care Cytokine IFNγIFNβ IFNGRIFNAR Early-phase clinical trials orPre-clinical Innate immune agonist Poly(I:C)-NTPoly(I:C)-TADU-S100SLR14 TLR3MDA5/TIG-I/TLR3STINGRIG-I Combination αPD-1 + IFNβ PD-1/IFNAR

To explore the expression of costimulatory molecule CD40 in thyroid tissue of Graves' disease patients and understand its immune pathogenetic significance. From January 2008 to December 2011, 8 patients undergoing partial thyroidectomy for Graves' disease (n = 3) or non-toxic goiter (n = 5) at Affiliated Suzhou Hospital of Nanjing Medical University and Third Affiliated Hospital of Soochow University were enrolled. Using the method of immunohistochemistry, the expression of CD40 was detected in their thyroid tissues. Variation in CD40 expression on thyroid follicular (TFC) in primary cultures was analyzed in the absence (no stimulation group) or presence of inflammatory cytokines including interleukin interferon-γ (IFN-γ) (IFN-γ stimulation group), interferon-6 (IL-6) (IL-6 stimulation group)and tumor necrosis factor (TNF)-α (TNF-α stimulation group) with flow cytometry. IFN-γ-stimulated TFC were cultured with agonist CD40 monoclonal antibody (5C11) (IFN-γ + CD40 group) or isotypic mouse IgG (mIgG) antibody (IFN-γ + mIgG group). And the proliferation of TFC was assessed by 3-(4,5)-dimethyl-thiazolyl-3,5-di-phenytetrazoliumromide (MTT) assays for each donor. The production of free triiodothyronine (FT3) and free thyroxine (FT4) and the release of thyroglobulin (Tg) were measured with radioimmunoassays. The expression of CD40 on infiltrated lymphocytes and TFC were detected in Graves' disease but not in non-toxic goiter patient tissues. Compared with no stimulation group (23.7% ± 7.3%), the expression of CD40 on TFC increased in IFN-γ stimulation group (86.4% ± 4.6%), IL-6 stimulation group (90.0% ± 4.2%) and TNF-α stimulation group (87.3% ± 4.2%). Compared with the IFN-γ + mIgG group (0.75 ± 0.06), the TFC proliferation of IFN-γ + CD40 group (1.14 ± 0.14) significantly increased (P < 0.01). The levels of FT3, FT4 and Tg secretion of IFN-γ + CD40 group were (1.10 ± 0.15) pmol/L, (0.80 ± 0.14) pmol/L and (30.23 ± 1.60) µg/L respectively. They were all significantly increased compared with the IFN-γ + mIgG group, of which the FT3, FT4 and Tg production were (0.76 ± 0.07) pmol/L, (0.63 ± 0.09) pmol/L and (21.37 ± 3.22) µg/L respectively (all P < 0.05). CD40 is abnormally expressed in thyroid tissue of Graves' disease patients. And its costimulatory signal may take part in the immunopathologic mechanism of Graves' disease.

Merkel cell carcinoma: an unusual case with SOX10 nuclear staining

The hypothesis of the cancer stem cell (CSC) suggests that neoplastic clones are maintained by a rare fraction of tumor cells with stem cell properties. CSCs could represent disseminated dormant tumor cells without clinical signs of tumor progression. We used a ret transgenic mouse spontaneous melanoma model, in which 25% of transgenic mice develop skin tumors with metastases in lymph nodes (mLN), liver, lungs and bone marrow (BM). Mice older than 20 weeks without macroscopic tumors contain in the BM tyrosinase related protein (TRP)-2-specific effector memory CD8+ T cells and show no further melanoma progression. This suggests a potential role of dormant tumor cells in the maintenance of memory CD8+ T cells. We found that TRP-2+CD133+ melanoma cells represent less than 1.5% of all cells in primary skin tumors and mLN. The majority of these cells were Ki67— suggesting thereby that these cells could remain in a dormant state. We found an increased expression of the major regulator for cell survival, self-renewal, and tumor growth, HIF-1a in TRP-2+CD133+ melanoma cells in large tumors in comparison with those in smaller tumors. To investigate whether TRP-2+CD133+ melanoma cells are disseminated in the BM of ret transgenic mice, we performed a triple immunofluorescence staining. We found that only 40% of mice without macroscopic tumors contained TRP-2+CD133+ melanoma cells in the BM. In contrast, all tumor bearing mice contained TRP-2+CD133+ melanoma cells. TRP-2+CD133+ melanoma cells were detected in 2 of 712 (0.238%) and 4 of 1285 (0.311%) disseminated melanoma cells in the BM of mice without and with macroscopic tumors, respectively. We confirmed the dormant state of TRP-2+CD133+ melanoma cells based on the negative expression of Ki67 and PCNA. Proteins p16 and p27, which are typically located in the nuclei of dormant cells, were found in the cytoplasmic compartment of TRP-2+CD133+ melanoma cells indicating their highly malignant phenotype. Investigating the interaction between memory CD8+ T cells with disseminated melanoma cells in the BM, we found that TRP-2+Ki67— melanoma cells were co-localized with memory CD8+ T cells both in mice without and with macroscopic tumors. The proportion of memory CD8+ T cells interacting with TRP-2+Ki67— melanoma cells was lower (less than 15%) in the BM of these mice. Quantitative analyses revealed that although certain IFN-g-producing CD8+ T cells interacted either with single TRP-2+ melanoma cells or the smallest cluster of melanoma cells (2-5 TRP-2+ cells), none of these T cells produced perforin. Only two TRP-2-specific CD8+ T cells produced perforin, but none of them were co-localized either with TRP-2+ melanoma cells or TRP-2+CD133+ melanoma cells. Furthermore, memory CD8+ T cells located within the large cluster of 50 TRP-2+ melanoma cells were unable to produce both perforin and IFN-g. These findings suggest that tumor microenvironment might neutralize CD8+ T cell reactivity. In conclusion, our data demonstrate the existence of a subpopulation of CD133+ melanoma cells in ret transgenic mice. Dormant TRP-2+ melanoma cells are able to interact with CD8+ T cells in the BM of tumor-bearing mice.

A representative poly(beta-amino ester) (PbAE) with biodegradable and pH-sensitive properties was used to formulate a nanoparticle-based dosage form for tumor-targeted paclitaxel delivery. The polymer undergoes rapid dissolution when the pH of the medium is less than 6.5 and hence is expected to release its contents at once within the acidic tumor microenvironment and endo/lysosome compartments of cells. PbAE nanoparticles were prepared by solvent displacement method and characterized for particle size, charge, and surface morphology. Pluronic F-108, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), was blended with PbAE to induce surface modification of the nanoparticles. In vitro cellular uptake of tritiated [(3)H]-paclitaxel in solution form and as a nanoparticulate formulation was studied in MDA-MB-231 human breast adenocarcinoma cells grown in 12-well plates. We also examined the intracellular degradation pattern of the formulations within the cells by estimating the drug release profile. Cytotoxicity assay was performed on the formulations at different doses and time intervals. Nanoparticles prepared from poly(epsilon-caprolactone) (PCL) that do not display pH-sensitive release behavior were used as control. Spherical nanoparticles having positive zeta potential ( approximately 40 mV) were obtained in the size range of 150-200 nm with PbAE. The PEO chains of the Pluronic were well-anchored within the nanomatrix as determined by electron spectroscopy for chemical analysis (ESCA). The intracellular accumulation of paclitaxel within tumor cells was significantly higher when administered in the nanoparticle formulations as compared to aqueous solution. Qualitative fluorescent microscopy confirmed the rapid release of the payload into the cytosol in the case of PbAE nanoparticles, while the integrity of the PCL nanoparticles remained intact. The cytotoxicity assay results showed significantly higher tumoricidal activity of paclitaxel when administered in the nanoparticle formulations. The cell-kill effect was maximal for paclitaxel-loaded PbAE nanoparticles when normalized with respect to intracellular drug concentrations. Thus, PEO-modified PbAE nanoparticles show tremendous potential as novel carriers of cytotoxic agents for achieving improved drug disposition and enhanced efficacy.

The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 μg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 μg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs‐loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 μg/ml and clear cytotoxicity at concentrations higher than 100 μg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.