Targeting CSPG4 for isolation of melanoma cell-derived exosomes from body fluids.

This manuscript describes the functional properties of the exosomes released from melanoma cells. It details the characteristics of the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4), which is used as a marker to separate exosomes released by melanoma cells from exosomes released by nonmalignant cells. The results are discussed in view of the potential role of melanoma cell-derived exosomes in the escape of malignant cells from the host's immune system.

Immunotherapeutics for head and neck squamous cell carcinoma stem cells.

Cancer stem cell (CSC)-related therapy resistance has become a new obstacle to the successful application of cancer treatment and head and neck squamous cell carcinoma (HNSCC) is no exception to this finding. Head and neck squamous cell carcinoma is highly immune-suppressive, and recently the immune suppression and invasion of HNSCC-CSCs have been characterized. These characteristics have received research and clinical attention because they would enable the stratification of patients into specific cancer subtypes and, consequently, the establishment of new therapeutic approaches with improved efficacy. This review discusses the feasibility of CSC-targeted strategies and their incorporation with nanotechnology to improve the efficacy of cancer immunotherapy.

Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

HLA-G 2012 conference: the 15-year milestone update.

The non-classical human leukocyte antigen (HLA) Class I molecule HLA-G is best known for its tolerogenic function at the maternal-fetal interface, where it protects the fetus from destruction by the immune system of its mother. Yet, HLA-G has been the topic of intense investigations and its functions reach much further than originally believed. International conferences on HLA-G have taken place every 3 years since 1998, and the Sixth International Conference on HLA-G, that took place in Paris in July 2012. It counted 180 attendees from 28 countries, 35 speakers in plenary sessions, and 63 presentations of research in symposia and poster sessions, bringing new insight in HLA-G research. Here we summarize the major advances on the function and nature of HLA-G molecule that were reported, with particular interest on the findings in new mechanisms of action through regulatory cells, its relevance in cancer as well as in the molecular structure and functions of HLA-G, which are key for its clinical application.

Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level.

BACKGROUND: The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients. METHODS: High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45(+) cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA(+), CD45(-), MART-1/gp100(+)). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis. RESULTS: CTC (HMW-MAA(+), CD45(-), MART-1/gp100(+)) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient. CONCLUSION: Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.

Cross-Instrument Comparison of MapCam and OVIRS on OSIRIS-REx.

Two of the instruments onboard the OSIRIS-REx spacecraft, the MapCam color imager and the OVIRS visible and infrared spectrometer, observed the surface of asteroid (101955) Bennu in partially overlapping wavelengths. Significant scientific advances have been enabled by using data from these two instruments in tandem, but a robust statistical understanding of their relationship is needed for future analyses to cross-compare their data as accurately and sensitively as possible. Here we present a cross-instrument comparison of data acquired by MapCam and OVIRS, including methods and results for all global and site-specific observation campaigns in which both instruments were active. In our analysis, we consider both the absolute radiometric offset and the relative (normalized) variation between the two instruments; we find that both depend strongly on the photometric and instrumental conditions during the observation. The two instruments have a large absolute offset (>15%) due to their independent radiometric calibrations. However, they are very consistent (relative offset as low as 1%) when each instrument's response is normalized at a single wavelength, particularly at low phase angles where shadows on Bennu's rough surface are minimized. We recommend using the global datasets acquired at 12:30 pm local solar time for cross-comparisons; data acquired at higher phase angles have larger uncertainties.

Selective histocompatibility leukocyte antigen (HLA)-A2 loss caused by aberrant pre-mRNA splicing in 624MEL28 melanoma cells.

Histocompatibility leukocyte antigen (HLA)-A2 is used as a restricting element to present several melanoma-associated antigen (MAA)-derived peptides to cytotoxic T lymphocytes (CTLs). HLA-A2 antigen is selectively lost in primary melanoma lesions and more frequently in metastases. Only scanty information is available about the molecular mechanisms underlying this abnormality, in spite of its potentially negative impact on the clinical course of the disease and on the outcome of T cell-based immunotherapy. Therefore, in this study we have shown that the selective HLA-A2 antigen loss in melanoma cells 624MEL28 is caused by a splicing defect of HLA-A2 pre-mRNA because of a base substitution at the 5' splice donor site of intron 2 of the HLA-A2 gene. As a result, HLA-A2 transcripts are spliced to two aberrant forms, one with exon 2 skipping and the other with intron 2 retention. The latter is not translated because of an early premature stop codon in the retained intron. In contrast, the transcript with exon 2 skipping is translated to a truncated HLA-A2 heavy chain without the alpha(1) domain. Such a polypeptide is synthesized in vitro but is not detectable in cells, probably because of the low steady state level of the corresponding mRNA and the low translation efficiency. These results indicate that a single mutational event in an HLA class I gene is sufficient for loss of the corresponding allele. This may account, at least in part, for the high frequency of selective HLA class I allele loss in melanoma cells. Our conclusion emphasizes the need to implement active specific immunotherapy with a combination of peptides presented by various HLA class I alleles. This strategy may counteract the ability of melanoma cells with selective HLA class I allele loss to escape from immune recognition.

DR (Ia-like) antigens on human melanoma cells. Serological detection and immunochemical characterization.

11 cultured human melanoma cell lines were tested for the expression of DR antigens by using specific allo- and xenoantisera in an indirect rosette microassay. Four of these melanoma cell lines expressed DR antigens, but in lower amounts than expressed on cultured human B-lymphoid cells. Rabbits injected with the DR-positive melanoma cells produced antibodies that were serologically and immunochemically reactive with B-cell-derived DR antigens. Immunochemical studies indicate that melanoma cell-derived DR antigens have a two-chain structure with 34,000 and 27,000 mol wt components. The melanoma cell-derived DR beta-chain at 27,000 mol wt is slightly smaller than that of the Victor cell DR beta-chain whose mol wt is 29,000.

Interaction of histocompatibility (HL-A) antibodies and complement with synchronized human lymphoid cells in continuous culture.

The interaction of histocompatibility (HL-A) antibodies and complement with synchronized human lymphoid cells in continuous culture has been investigated. The sensitivity of cultured lymphoid cells to HL-A antibody-mediated lysis in the cytotoxic test, the extent of activation of the complement system, the degree to which labeled complement components are bound, and the ability of these cells to absorb HL-A alloantibodies do not vary significantly during the cell growth cycle. The constancy of histocompatibility antigen expression throughout the growth cycle of cultured cells suggests that these cell surface markers are an essential part of membrane cytoarchitecture and could well play a critical role in determining the normal function of the cell membrane.

Variation in susceptibility of a human lymphoid cell line to immune lysis during the cell cycle. Lack of correlation with antigen density and complement binding.

Cultured human lymphoid cells RPMI 8866 at different stages of their growth cycle vary in their susceptibility to lysis by rabbit, human, and guinea pig complement activated by HL-A antibodies or heterologous antibodies directed to membrane antigens; cells in G(1) phase are the least sensitive to lysis. To investigate the cause of differential susceptibility of cells RPMI 8866 to lysis, the expression of HL-A determinants and the ability of cells to react with complement were investigated. No change was detected in the density of HL-A antigens on RPMI 8866 cells in synchronous growth as determined by quantitative microabsorption assays, isotopic antiglobulin tests and yields of soluble HL-A antigens. Cells did not vary during the growth cycle in their ability to interact with complement components and in their capacity to activate the complement system through the classical or alternate pathway. These data suggest that variability in lytic susceptibility is due to changes in the structure of the cell membrane or in its ability to repair complement induced damage at certain intervals during the cell cycle. Therefore, this cell line constitutes a useful model to investigate the final steps of the cytolytic reaction.

Bright carbonate veins on asteroid (101955) Bennu: Implications for aqueous alteration history.

The composition of asteroids and their connection to meteorites provide insight into geologic processes that occurred in the early Solar System. We present spectra of the Nightingale crater region on near-Earth asteroid Bennu with a distinct infrared absorption around 3.4 micrometers. Corresponding images of boulders show centimeters-thick, roughly meter-long bright veins. We interpret the veins as being composed of carbonates, similar to those found in aqueously altered carbonaceous chondrite meteorites. If the veins on Bennu are carbonates, fluid flow and hydrothermal deposition on Bennu's parent body would have occurred on kilometer scales for thousands to millions of years. This suggests large-scale, open-system hydrothermal alteration of carbonaceous asteroids in the early Solar System.

Expression of C4 on human lymphoid cells and possible involvement in immune recognition phenomena.

Immunochemical studies revealed the presence of the fourth component of complement (C4) on surfaces of human lymphoid cells. Antiserums to C4 inhibited the mixed lymphocyte reaction and the mitogenic response to phytohemagglutinin, suggesting a role for membrane-associated C4 in the afferent phase of immune recognition phenomena.

Tumor escape mechanisms: potential role of soluble HLA antigens and NK cells activating ligands.

The crucial role played by human leukocyte antigen (HLA) antigens and natural killer (NK)-cell-activating ligands in the interactions of malignant cells with components of the host's immune system has stimulated interest in the characterization of their expression by malignant cells. Convincing evidence generated by the immunohistochemical staining of surgically removed malignant lesions with monoclonal antibodies recognizing HLA antigens and NK-cell-activating ligands indicates that the surface expression of these molecules is frequently altered on malignant cells. These changes appear to have clinical significance because in some types of malignant disease they are associated with the histopathological characteristics of the lesions as well as with disease-free interval and survival. These associations have been suggested to reflect the effect of HLA antigen and NK-cell-activating ligand abnormalities on the interactions of tumor cells with antigen-specific cytotoxic T lymphocytes (CTL) and with NK cells. Nevertheless, there are examples in which disease progresses in the face of appropriate HLA antigen and/or NK-cell-activating ligand as well as tumor antigen expression by malignant cells and of functional antigen-specific CTL in the investigated patient. In such scenarios, it is likely that the tumor microenvironment is unfavorable for CTL and NK cell activity and contributes to tumor immune escape. Many distinct escape mechanisms have been shown to protect malignant cells from immune recognition and destruction in the tumor microenvironment. In this article, following the description of the structural and functional characteristics of soluble HLA antigens and NK-cell-activating ligands, we will review changes in their serum level in malignant disease and discuss their potential role in the escape mechanisms used by tumor cells to avoid recognition and destruction.

Production and characterization of monoclonal antibodies against major histocompatibility complex class I chain-related gene A.

Major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand for the activating immunoreceptor natural killer group 2D (NKG2D), is expressed on stressed cells such as tumor cells. Study of expression of this molecule on tumor cells and patients' sera is useful to define patients' stages leading to proper selection of therapy. In this study, mouse anti-MICA monoclonal antibodies (mAbs) were produced by DNA immunization using a gene gun. Screening of anti-MICA-producing mouse and hybridomas were performed by immunoblot and cell enzyme-linked immunosorbent assay (ELISA) against MICA-positive HeLa and -negative Me1386 cell lines. MAbs were characterized against MICA-positive and -negative cell lines by immunoblot, cell ELISA and flow cytometry. The mAbs were also characterized for locus and allele specificities of MICA and MHC class I chain-related gene B (MICB) as well as for their ability to stain formalin-fixed paraffin-embedded tissues by immunohistochemistry. Although all mouse immune sera were positive with MICA-positive cells by both immunoblot and cell ELISA methods, some hybridomas were positive only with one method. The mAbs had diverse specificities to detect MICA and MICB and different abilities to stain formalin-fixed paraffin-embedded tissues. Thus, DNA immunization by gene gun is an effective method to generate immune mice for the production of mAbs with a variety of specificities against native and denatured forms of MIC proteins.

Regulation of high molecular weight-melanoma associated antigen (HMW-MAA) gene expression by promoter DNA methylation in human melanoma cells.

The human high molecular weight-melanoma associated antigen (HMW-MAA) is a membrane-bound chondroitin sulfate proteoglycan that is variably expressed in a high percentage of melanoma cell lines and tumors. Since the mechanism(s) regulating HMW-MAA expression has(ve) not been defined, in this study, we have examined whether promoter DNA methylation regulates the level of HMW-MAA expression. In melanoma cell lines, the level of HMW-MAA mRNA and protein expression is coordinately regulated, implicating a transcriptional control mechanism. Consistent with a role for regulation by DNA methylation, we have found that a dense CpG island flanks the human HMW-MAA gene transcriptional start site. Methylation-specific PCR and sodium bisulfite DNA sequencing analyses indicate that the HMW-MAA promoter is heavily methylated in melanoma cell lines, melanoma lesions and normal lymphocytes that do not express HMW-MAA; in contrast, the HMW-MAA promoter is not methylated in melanoma cell lines and tumors that express this antigen. In addition, HMW-MAA expression is markedly induced in HMW-MAA-negative melanoma cell lines by incubation with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. In summary, our results establish DNA methylation as a key regulator of HMW-MAA expression by human melanoma cells. This information represents a useful background to optimize immunotherapeutic strategies targeting HMW-MAA.

Murine antiidiotypic monoclonal antibodies that bear the internal image of HLA-DR allospecificities.

Hybridization of murine myeloma cells P3-X63-Ag8.653 with splenocytes from a BALB/c mouse immunized with the syngeneic anti HLA-DR1,4,w6,w8,w9 MAb AC1.59 resulted in the development of 108 hybridomas secreting antiidiotypic antibodies. 100 of them inhibited the binding of MAb AC1.59 to target cells. Detailed analysis of the antiidiotypic MAb F5-444, F5-830, F5-963, F5-1126, F5-1336, and F5-1419 showed that all of them recognize idiotopes within or spatially close to the antigen combining site of MAb AC1.59. In cross-blocking experiments, the six antiidiotypic MAbs cross-blocked each other. It is likely that the six MAbs recognize spatially close, but not identical idiotopes because they elicited antiantiidiotypic antibodies of different or similar, but not identical specificity and differ in their ability to elicit anti-HLA class II antibodies. The latter, which were found only in sera from BALB/c mice immunized with antiidiotypic MAb F5-444 and F5-830, mimic the specificity of MAb AC1.59 and express the idiotope defined by the immunizing antiidiotypic MAb. These results indicate that the MAb F5-444 and F5-830 are antiidiotypes beta and the remaining four are antiidiotypes gamma.

Reduction in susceptibility to natural killer cell-mediated lysis of human FO-1 melanoma cells after induction of HLA class I antigen expression by transfection with B2m gene.

Induction of HLA class I antigens on cultured melanoma cells FO-1 after transfection with a human or a mouse B2m gene was associated with a statistically significant reduction in their susceptibility to natural killer (NK) cell-mediated lysis. These results indicate that the structural differences between human and mouse beta 2-mu do not abolish the ability of the HLA class I molecular complex to modulate NK cell-mediated lysis of melanoma cells FO-1. The role of HLA class I antigens in the phenomenon is corroborated by the ability of anti-HLA class I MAb to enhance, although to a different extent, the susceptibility of transfected FO-1 cells to NK cell-mediated lysis. Gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) significantly reduced the susceptibility to NK cell-mediated lysis of transfected FO-1 cells. Surprisingly, TNF-alpha reduced the extent of lysis more than IFN-gamma, although the latter cytokine enhanced HLA class I antigen expression more than the former one. This finding, in conjunction with a reduction in the susceptibility to NK cell-mediated lysis of untransfected FO-1 cells incubated with IFN-gamma or TNF-alpha, suggests that the two cytokines reduce NK cell-mediated lysis of transfected cells by modulating not only the expression of HLA class I antigens, but also that of other structures. Induction of HLA class I antigens and their modulation with IFN-gamma did not affect the susceptibility to lymphokine-activated killer (LAK) cell-mediated lysis of transfected FO-1 cells. Characterization of the molecular mechanism(s) underlying abnormalities in HLA class I antigen expression by melanoma cells and of the role of these molecules in the interactions of melanoma cells with various types of effector cells may suggest novel immunotherapeutic approaches to melanoma.

Utilization of cultured human lymphoid cells for detection of humoral sensitization in prospective recipients of kidney transplants.

Prospective recipients of kidney transplants were tested for lymphocytotoxicity; from these we selected 102 sera that lacked cytotoxic antibodies against peripheral lymphocytes from at least 80 unrelated subjects. To detect humoral sensitization, we then reacted these with 17 cultured human lymphoid cell lines having different HL-A phenotypes. Cytotoxic antibodies reacting with these cultured cells were now detected in some of the sera. These antibodies were not directed against HL-A antigens, yet mediated lysis of target cells in the presence of rabbit but not of human or guinea pig complement. Furthermore, they activated the classical pathway of the rabbit complement system. Later, a significant association was found between occurrence of cytotoxic antibodies and rejection of the transplant. Thus, cultured human lymphoid cells, because of their great susceptibility to complement-mediated lysis, appear to be useful in detecting humoral sensitization in candidates for kidney grafts.

beta2-Microglobulin mutations, HLA class I antigen loss, and tumor progression in melanoma.

The potential negative impact of HLA class I antigen abnormalities on the outcome of T cell-based immunotherapy of melanoma has prompted us to investigate the mechanisms underlying lack of HLA class I antigen expression by melanoma cell lines Me18105, Me9923, and Me1386. Distinct mutations in the beta2-microglobulin (beta2m) gene were identified in each cell line which result in loss of functional beta2m. In Me18105 cells, an aberrant splicing mechanism caused by an A--> G point mutation in the splice acceptor site of intron 1 of the beta2m gene, deletes 11 bp from the beta2m mRNA creating a shift in the reading frame. In Me9923 cells a 14-bp deletion in exon 2 and in Me1386 cells a CT deletion in exon 1 of the beta2m gene produce a frameshift mutation. The beta2m gene mutations identified in Me18105, Me9923, and Me1386 cells were also detected in the surgically removed melanoma lesions from which the cell lines originated. Transfection of each melanoma cell line with a wild-type beta2m gene restored HLA class I antigen expression and, in Me18105 cells, recognition by Melan-A/MART-1-specific, HLA-A2-restricted cytotoxic T lymphocytes. Interestingly, the beta2m mutation present in Me9923 cells that were derived from a metastatic lesion was also found in the Me9923P cell line that originated from the autologous primary lesion. These data suggest that beta2m mutations in melanoma cells may be an early event in progression to the malignant phenotype.