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.