Rare osteosarcoma cell subpopulation protein array and profiling using imaging mass cytometry and bioinformatics analysis.

BACKGROUND: Single rare cell characterization represents a new scientific front in personalized therapy. Imaging mass cytometry (IMC) may be able to address all these questions by combining the power of MS-CyTOF and microscopy. METHODS: We have investigated this IMC method using < 100 to up to 1000 cells from human sarcoma tumor cell lines by incorporating bioinformatics-based t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis of highly multiplexed IMC imaging data. We tested this process on osteosarcoma cell lines TC71, OHS as well as osteosarcoma patient-derived xenograft (PDX) cell lines M31, M36, and M60. We also validated our analysis using sarcoma patient-derived CTCs. RESULTS: We successfully identified heterogeneity within individual tumor cell lines, the same PDX cells, and the CTCs from the same patient by detecting multiple protein targets and protein localization. Overall, these data reveal that our t-SNE-based approach can not only identify rare cells within the same cell line or cell population, but also discriminate amongst varied groups to detect similarities and differences. CONCLUSIONS: This method helps us make greater inroads towards generating patient-specific CTC fingerprinting that could provide an accurate tumor status from a minimally-invasive liquid biopsy.

Discovery of Cell-Surface Vimentin (CSV) as a Sarcoma Target and Development of CSV-Targeted IL12 Immune Therapy.

This chapter discusses a novel target of osteosarcoma (OS), cell-surface vimentin (CSV), and a novel generation of interleukin-12 (IL12), CSV-targeted IL12, for treating OS tumor metastasis. Vimentin is a known intracellular structural protein for mesenchymal cells but is also documented in tumor cells. Our recent study definitively revealed that vimentin can be translocated to the surface of very aggressive tumor cells, such as metastatic cells. This CSV property allows investigators to capture circulating tumor cells (CTCs) across any type of tumor, including OS. CTCs are known as the seeds of metastasis; therefore, targeting these cells using CSV is a logical approach for use in a metastatic OS setting. Interestingly, we found that the peptide VNTANST can bind to CSV when fused to the p40 subunit encoding the DNA of IL12. Systemic delivery of this CSV-targeted IL12 immune therapy inhibited OS metastasis and relapse in a mouse tumor model as detailed in this chapter. This CSV-targeted delivery of IL12 also reduced toxicity of IL12. In summary, this chapter details a novel approach for safe IL12 immune therapy via targeting CSV.

Cell surface vimentin-positive circulating tumor cell-based relapse prediction in a long-term longitudinal study of postremission neuroblastoma patients.

Neuroblastoma (NB) is a deadly childhood disease that carries a 50% chance of relapse for anyone in remission and similar level of 5-year survival. We investigated the value of our proprietary approach-cell surface vimentin (CSV) positive circulating tumor cells (CTC) to monitor treatment response and predict relapse in NB patients under remission in a Phase II long-term preventative clinical trial. We longitudinally analyzed peripheral blood samples from 93 patients for 27 cycles (~25 months) and discovered that the presence of CSV+ CTCs in the first two sequential samples (baseline, cycle 4 [month 3-4]) was a significant indicator of earlier relapse. We observed strong correlation between relapse-free survival (RFS) and lack of CSV+ CTCs in first 4 cycles of therapy (95%). There was sensitivity reaching 100% in predicting RFS in patients who had neither CSV+ CTCs nor MycN amplification. Of note, the low number of CSV+ CTCs seems equivalent to low tumor load because the prevention therapy difluoromethylornithine yields faster reduction of relapse risk when none or only 1-2 CSV+ CTCs (every 6 mL) are present in the blood samples compared to >3 CSV+ CTCs. To the best of our knowledge, this is the first study that directly observes CTCs in under remission NB patients for relapse prediction and the first to gather sequential CSV+ CTC data in any study in a long-term longitudinal manner.

CTC analysis: an update on technological progress.

There is a growing need for a more accurate, real-time assessment of tumor status and the probability of metastasis, relapse, or response to treatment. Conventional means of assessment include imaging and tissue biopsies that can be highly invasive, may not provide complete information of the disease's heterogeneity, and not ideal for repeat analysis. Therefore, a less-invasive means of acquiring similar information at greater time points is necessary. Liquid biopsies are samples of a patients' peripheral blood and hold potential of addressing these criteria. Ongoing research has revealed that a tumor can release circulating cells, genetic materials (DNA or RNA), and exosomes into circulation. These potential biomarkers can be captured in a liquid biopsy and analyzed to determine disease status. To achieve these goals, numerous technologies have been developed. In this review, we discuss both prominent and newly developed technologies for circulating tumor cell capture and analysis and their clinical impact.

Cell-surface vimentin-positive macrophage-like circulating tumor cells as a novel biomarker of metastatic gastrointestinal stromal tumors.

The clinical utility of circulating tumor cells (CTCs) has been investigated in numerous publications, but CTCs that express very typical immune cell markers have not been reported. Here we report a novel class of CTCs-CSV-positive macrophage-like CTCs (ML-CTCs). This nomenclature was based on the fact that this class of CTCs can be captured from blood samples of gastrointestinal stromal tumors (GISTs) patients using either the macrophage marker CD68 or our proprietary tumor-specific cell-surface vimentin (CSV) antibody 84-1; likewise, the captured ML-CTCs can be co-stained with both typical macrophage markers (CD14, CD68) and tumor cell markers (DOG-1, C-kit) but not CD45. Patients with metastatic GIST had significantly greater numbers of ML-CTCs than patients with localized GIST or cancer-free blood donors (P<0.0001). Unexpectedly, the classic CSV positive CTCs was abundant in metastatic disease but failed to predict GIST metastasis. Only CSV-positive ML-CTCs was able to serve as a solid and novel biomarker for prediction of metastatic risk in GIST patients.

Regulation of NKG2D+CD8+ T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism.

The natural killer (NK) group 2D (NKG2D) receptor, which displays on mouse and human NK cells, activates CD8+ T cells and small subsets of other T cells. NKG2D+CD8+ T cells play critical roles in both innate and adaptive immunity upon engagement with NKG2D ligands to eliminate tumor and infected cells. Despite the important role of NKG2D+CD8+ T cells in immune surveillance, the mechanisms of how NKG2D expression on CD8+ T cells is regulated remain poorly defined. We treated mouse and human CD8+ T cells with CD80 recombinant protein, plus a pharmacologic model with small molecular inhibitors to determine which signaling pathway leads to NKG2D regulation on CD8+T cells. This study revealed that CD28 activation gives rise to sustained NKG2D expression on both mouse and human CD8+ T cells in a signal transducer and activator of transcription 3 (STAT3) phosphorylation-dependent manner. Further, we found that CD28 activation stimulated sustained activation of the tyrosine kinase Lck, which recruits and triggers Janus kinase/STAT3 signaling to phosphorylate STAT3, and in turn increases NKG2D expression. Moreover, NKG2D induction on CD8+ T cells exerts cytolytic activity against target tumor cells in vitro, as well as significantly improves the antitumor therapeutic effects in vivo in an NKG2D-dependent manner. Taken together, these results elucidated a novel mechanism of NKG2D regulation by phosphorylated STAT3 (pSTAT3) on CD8+ T cells upon CD28 activation. This mechanism may shed light on the effectiveness of CD80-based, NKG2D-dependent antitumor immunotherapy.

S100B interaction with the receptor for advanced glycation end products (RAGE): a novel receptor-mediated mechanism for myocyte apoptosis postinfarction.

RATIONALE: Post-myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis. OBJECTIVE: This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model. METHODS AND RESULTS: In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations > or = 50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis. CONCLUSIONS: These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

Homologous recombination involving cox2 is responsible for a mutation in the cmS-specific mitochondrial locus of Petunia.

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exonl of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5' sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population.

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant.

Morphology, the extent of elimination of donor chromosomes and the organelle composition of highly asymmetric somatic hybrid plants between a interspecific tomato hybrid Lycopersicon esculentum x L. pennellii (EP) as donor and a Solarium melongena, eggplant (E), recipient, were studied. Morphologically, the somatic hybrids most resemble eggplant but, due to polyploidy, growth is slower relative to both fusion parents. The somatic hybrids produce flowers that are characterized by abnormal styles, stigmas and by anthers which do not produce pollen. Limited amounts of donor EP genomic DNA were found in the three somatic hybrid plants (H18-1, H18-2 and H18-3), by dot-blot hybridization with probe pTHG2, equivalent to 6.23,5.41, and 5.95% EP, respectively. These percentages translated to the presence of 3.59, 2.90 and 3.19 average-size EP chromosomes in plants H1 8-1,-2 and-3, respectively. RFLP determination of L. esculentum- and L. pennellii-specific chromosomes revealed that only fragments of eight to ten out of the 24 EP chromosomes (EP has 12 L. esculentum and 12 L. pennellii chromosomes) are present in the asymmetric somatic hybrid plants. Loci of L. esculentum and L. pennellii were evenly represented in plants H18-1, -2, and -3: four to five from L. esculentum and four to five from L. pennellii. All somatic hybrid plants retained locus TG22, chromosome 4, from both EP species. Although the regeneration of plants, H18-1, -2 and-3 was from one callus, loci TG31 and TG79 of L. esculentum chromosome 2 and L. pennellii chromosome 9, respectively, were missing in hybrid plant H18-1. The three somatic hybrid plants all had chloroplast DNA fragments specific for S. melongena. The mitochondrial genome (mtDNA) in the asymmetric somatic hybrids showed predominantly the pattern of eggplant; however, some eggplant-specific polymorphic bands were not present in the three plants.

Involvement of two different urf-s related mitochondrial sequences in the molecular evolution of the CMS-specific S-Pcf locus in petunia.

In petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, Pcf, contains parts of the atp9 and coxII genes and an unidentified reading frame, urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. The nad3 and rps12 sequences included in the S-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy of nad3 and rps12 had been detected on the physical map of the main mt genome. The origin of the urf-s sequence and the molecular events leading to the formation of the chimeric S-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related to urf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of the S-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of these urf-s related sequences (showing 100% homology to urf-s and termed orf-h) is located on a sublimon. An additional, low-homology urf-s related sequence (Rf-1) is shown to be located on the main mt genome 5' to the nad3 gene. It is, thus, suggested that the sequence of events leading to the generation of the S-Pcf locus might have involved introduction of the orf-h sequence, via homologous recombination, into the main mt genome 5' to nad3 at the region where the Rf-1 sequence is located.

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia.

Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5-7.0 for the membrane-bound fraction and between 4.5-7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg(2+). At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20-50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.

Detection of an open reading frame related to the CMS-associated urf-s in fertile Petunia lines and species and in other fertile Solanaceae species.

In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.

Interaction of the mitochondrial S-Pcf locus for cytoplasmic male sterility in Petunia with multiple fertility-restoration genes in somatic hybrid plants.

The Cytoplasmic Male Sterility (CMS)-associated region in Petunia, the S-Pcf locus, was defined by the analysis of recombinant mitochondrial genomes of somatic hybrid plants resulting from a fusion of protoplasts from CMS and fertile lines. The presence of the S-Pcf locus was shown to correlate with the CMS trait in stable somatic hybrids and in other CMS Petunia lines. A small population of unstable, sterile somatic hybrids was also generated in this fusion, most of which underwent cytoplasmic segregation in subsequent generations. Stable revertants of such sterile somatic hybrids were shown to lose the S-Pcf locus. In this paper we present a molecular and genetic analysis of unstable progenies of an unstable, sterile somatic hybrid plant derived from the same fusion experiment. Both male-sterile and fertile progenies of this somatic hybrid plant have shown continuous segregation of fertile and male-sterile progenies. All segregants in this line contained, and transcribed, the S-Pcf locus. Genetic analysis indicated the presence of various levels of multiple nuclear fertility-restoration genes in this group of progenies. These findings consolidate the association between the S-Pcf locus and the CMS trait in Petunia. It also shows that the restoration of fertility by the multiple nuclear gene system does not affect the transcription of the S-Pcf locus and that the presence of an intact S-Pcf locus is necessary in order to maintain the potential sterility in the cytoplasm.

Differences in amino acid transport in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia.

Two pairs of isonuclear lines of cytoplasmic male-sterile (CMS) and fertile (F) petunia cells grown in suspension culture in the presence or absence of amino acid sources were examined for uptake of 11 amino acids and adenosine. Cells from CMS lines exhibited a significant lower rate of uptake than F cells. These differences, for various amino acids, are a result of lower affinity (high Km) values and of lower maximal velocities. Although the uptake of most of the amino acids examined was affected by the availability of energy in the cell, the differences in uptake seem to be less dependent on the energy status of the cell.

Attempts to detect extra genomial factors in cytoplasmic male-sterile petunia lines.

In the present study we examined the possibility that viruses, viroids or dsRNA are associated with cytoplasmic male sterile (cms) petunia. The assumption was made that if viruses or viroids were present, the treatments for elimination of viruses and viroids would produce "healthy" fertile plants. Male sterile plants were subjected to heat and cold treatments for 10 weeks and/ or for 5 months, after which apical meristems were isolated and cultured with the addition of antiviral factors. The mother plants, the regenerated plants and their progeny were sterile. These treatments did not affect sterility in sterile plants or the fertility of fertile plants. No dsRNA was found in cms petunia by gel electrophoresis. Thus, our data suggest that there are no viruses, viroids or dsRNA associated with cms petunia. Our data are in agreement with recent data, which suggests that the mitochondrial DNA is the site of the cytoplasmic male sterile gene in petunia.

Loss of CMS-specific mitochondrial DNA arrangement in fertile segregants of Petunia hybrids.

The progeny of somatic hybrid Petunia plants derived from the fusion of a male-fertile line and a cytoplasmic male-sterile (cms) line were examined. Male-fertile progeny derived from three different male-sterile somatic hybrid plants did not exhibit the mitochondrial DNA (mtDNA) arrangement which has previously been correlated with cms in Petunia. The cms-associated mtDNA arrangement was present in the male-sterile predecessors of these fertile revertants. Thus, it is concluded that the loss of this mtDNA arrangement is associated with reversion to fertility in the progeny of the unstable somatic hybrid petunia plants.

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids.

We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped.Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.

An in vitro Screening for Tomato Genotypes Exhibiting Efficient Shoot Regeneration.

Over 100 genotypes of tomato (Lycopersicon esculentum, wild relatives and suspected interspecific crosses) were assayed for shoot regeneration from in vitro cultured hypocotyl segments. Expiants incubated for 8 weeks in Murashige and Skoog medium supplemented with 2% glucose, 1 mg zeatin and 0.02 mg · l(-1) IAA, exhibited the best overall shoot regeneration. A wide variability in this response was observed, and approximately one-fourth (23/89) of the L. esculentum tested genotypes exhibited relatively high differentiation capabilities (>5 shoots per expiant).

Induction of high mitotic index in Petunia suspension cultures by sequential treatment with aphidicolin and colchicine.

Significantly higher than normal mitotic index (MI) values were induced in Petunia cell suspensions following treatments with colchicine, aphidicolin, drastic medium replacement, or a sequential application of aphidicolin and colchicine. This last treatment yielded the highest MI values: cells incubated with 30 µg/ml aphidicolin for 18 h, then cultured in drug-free medium for 8 h and finally exposed to 0.1% colchicine for 8 additional hours exhibited MI of 62.8% and 65.7% respectively, for the two cell lines in study.

Improved efficiency of plant regeneration from protoplasts of eggplant Solanum melongena L.

Eggplant (Solanum melongena L.) mesophyll protoplasts were obtained from in vitro growing plants of line 410 and cv. 'Classic'. Relatively high (15%) plating efficiency was achieved using petri dishes with alternate quadrants containing reservoir medium (R medium + 1% activated charcoal) and culture medium. Shoot regeneration occurred within 6 weeks following initiation of protoplast culture.