Evidence for 2-Methoxyestradiol-Mediated Inhibition of Receptor Tyrosine Kinase RON in the Management of Prostate Cancer.

2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

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.

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.

IGF-IR signaling in epithelial to mesenchymal transition and targeting IGF-IR therapy: overview and new insights.

The insulin-like growth factor-I (IGF-I) signaling induces epithelial to mesenchymal transition (EMT) program and contributes to metastasis and drug resistance in several subtypes of tumors. In preclinical studies, targeting of the insulin-like growth factor-I receptor (IGF-IR) showed promising anti-tumor effects. Unfortunately, high expectations for anti-IGF-IR therapy encountered challenge and disappointment in numerous clinical trials. This review summarizes the regulation of EMT by IGF-I/IGF-IR signaling pathway and drug resistance mechanisms of targeting IGF-IR therapy. Most importantly, we address several factors in the regulation of IGF-I/IGF-IR-associated EMT progression that may be potential predictive biomarkers in targeted therapy.

Recepteur d'origine nantais (RON), more than a kinase: Role in castrate-resistant prostate cancer.

Prostate cancer (PCA) is the second leading cause of cancer-related deaths in men in the United States. It is natural for a hormone-driven malignancy such as prostate cancer that androgen deprivation therapy (ADT) would be the preferred treatment for clinical disease management. However, after initial treatment response a vast majority of patients develop metastatic castrate-resistant prostate cancer (CRPC), which is fatal. While great headway has been made to understand the possible mechanisms that drive castrate-resistant disease, a bonafide cure remains elusive. Reactivation of androgen receptor (AR) signaling partly contributes to the emergence of CRPC. Here we briefly examine some of the known mechanisms of AR reactivation including intratumoral synthesis of androgens, modulation of AR coregulators, and AR variants with constitutive activity as well as activation of receptor tyrosine kinases. We primarily focus on the emerging dual function of the receptor tyrosine kinase (recepteur d'origine nantais; RON) as a traditional tyrosine kinase and transcription factor. We further discuss activation of RON as an alternate mechanism in the development of CRPC and available therapeutic approaches for clinical management of CRPC by combined inhibition of RON and AR.

Palmatine inhibits growth and invasion in prostate cancer cell: Potential role for rpS6/NFκB/FLIP.

Novel agents are desperately needed for improving the quality of life and 5-year survival to more than 30% for metastatic castrate-resistant prostate cancer. Previously we showed that Nexrutine, Phellodendron amurense bark extract, inhibits prostate tumor growth in vitro and in vivo. Subsequently using biochemical fractionation we identified butanol fraction contributes to the observed biological activities. We report here that palmatine, which is present in the butanol fraction, selectively inhibits growth of prostate cancer cells without significant effect on non-tumorigenic prostate epithelial cells. By screening receptor tyrosine kinases in a protein kinase array, we identified ribosomal protein S6, a downstream target of p70S6K and the Akt/mTOR signaling cascade as a potential target. We further show that palmatine treatment is associated with decreased activation of NFκB and its downstream target gene FLIP. These events led to inhibition of invasion. Similar results were obtained using parent extract Nexrutine (Nx) suggesting that palmatine either in the purified form or as one of the components in Nx is a potent cytotoxic agent with tumor invasion inhibitory properties. Synergistic inhibition of rpS6/NFκB/FLIP axis with palmatine may have therapeutic potential for the treatment of prostate cancer and possibly other malignancies with their constitutive activation. These data support a biological link between rpS6/NFκB/FLIP in mediating palmatine-induced inhibitory effects and warrants additional preclinical studies to test its therapeutic efficacy.

Automated Capture and Analysis of Circulating Tumor Cells in Pediatric, Adolescent and Young Adult Patients with Central Nervous System Tumors.

Tumors of the central nervous system (CNS) are the most common and lethal childhood malignancy. Detection of residual disease and longitudinal monitoring of treatment response in patients are challenging and rely on serial imaging. This current standard of care fails to detect microscopic disease or provide molecular characteristics of residual tumors. As such, there is dire need for minimally invasive liquid biopsy techniques. We have previously shown the high specificity of using cell surface vimentin (CSV) to identify circulating tumor cells (CTCs) from patients bearing various types of cancers. Here, we describe the first report of CTCs captured from peripheral blood samples in 58 pediatric CNS tumor patients. In this study, we used a CSV-coated cell capture chip, the Abnova CytoQuest automated CTC isolation system, to boost the CTC capture from pediatric patients with CNS tumors. We successfully isolated CTCs in six glioma patients using immunostaining of histone H3 lysine27-to-methionine (H3K27M) mutations which are highly expressed by this tumor. We show that CSV is a viable marker for CNS CTC isolation and that this is a feasible method for detecting microscopic disease. Larger-scale studies focusing on CTCs in pediatric CNS tumors to explore their diagnostic and prognostic value are warranted.

Prognostic Value of Cell-Surface Vimentin-Positive CTCs in Pediatric Sarcomas.

BACKGROUND: Despite advances in care, the 5 year overall survival for patients with relapsed and or metastatic sarcoma remains as low as < 35%. Currently, there are no biomarkers available to assess disease status in patients with sarcomas and as such, disease surveillance remains reliant on serial imaging which increases the risk of secondary malignancies and heightens patient anxiety. METHODS: Here, for the first time reported in the literature, we have enumerated the cell surface vimentin (CSV+) CTCs in the blood of 92 sarcoma pediatric and adolescent and young adult (AYA) patients as a possible marker of disease. RESULTS: We constructed a ROC with an AUC of 0.831 resulting in a sensitivity of 85.3% and a specificity of 75%. Additionally, patients who were deemed to be CSV+ CTC positive were found to have a worse overall survival compared to those who were CSV+ CTC negative. We additionally found the use of available molecular testing increased the accuracy of our diagnostic and prognostic tests. CONCLUSIONS: Our findings indicate that CSV+ CTCs have prognostic value and can possibly serve as a measure of disease burden.

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.

EMT circulating tumor cells detected by cell-surface vimentin are associated with prostate cancer progression.

Recent advances in the field of circulating tumor cells (CTC) have shown promise in this liquid biopsy-based prognosis of patient outcome. However, not all of the circulating cells are tumor cells, as evidenced by a lack of tumor-specific markers. The current FDA standard for capturing CTCs (CellSearch) relies on an epithelial marker and cells captured via CellSearch cannot be considered to have undergone EMT. Therefore, it is difficult to ascertain the presence and relevance of any mesenchymal or EMT-like CTCs. To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma, breast, and colon cancer. Here we studied peripheral blood samples of 48 prostate cancer (PCA) patients including hormone sensitive and castration resistant sub-groups. Blood samples were analyzed for three different properties including our own CSV-based CTC enumeration (using 84-1 mAb against CSV), CellSearch-based epithelial CTC counts, and serum prostate-specific antigen (PSA) quantification. Our data demonstrated that in comparison with CellSearch, the CSV-based method had greater sensitivity and specificity. Further, we observed significantly greater numbers of CTCs in castration resistant patients as measured by our CSV method but not CellSearch. Our data suggests CSV-guided CTC enumeration may hold prognostic value and should be further validated as a possible measurement of PCA progression towards the deadly, androgen-independent form.

Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells.

Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as "seed" cells for tumor metastasis. The utility of CTCs in clinical applications for sarcoma is not fully investigated, partly owing to the necessity for fresh blood samples and the lack of a CTC-specific antibody. To overcome these drawbacks, we developed a technique for sarcoma CTCs capture and detection using cryopreserved peripheral blood mononuclear cells (PBMCs) and our proprietary cell-surface vimentin (CSV) antibody 84-1, which is specific to tumor cells. This technique was validated by sarcoma cell spiking assay, matched CTCs comparison between fresh and cryopreserved PBMCs, and independent tumor markers in multiple types of sarcoma patient blood samples. The reproducibility was maximized when cryopreserved PBMCs were prepared from fresh blood samples within 2 h of the blood draw. In summary, as far as we are aware, ours is the first report to capture and detect CTCs from cryopreserved PBMCs. Further validation in other types of tumor may help boost the feasibility and utility of CTC-based diagnosis in a centralized laboratory.

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.

Potential role of nuclear PD-L1 expression in cell-surface vimentin positive circulating tumor cells as a prognostic marker in cancer patients.

Although circulating tumor cells (CTCs) have potential as diagnostic biomarkers for cancer, determining their prognostic role in cancer patients undergoing treatment is a challenge. We evaluated the prognostic value of programmed death-ligand 1 (PD-L1) expression in CTCs in colorectal and prostate cancer patients undergoing treatment. Peripheral blood samples were collected from 62 metastatic colorectal cancer patients and 30 metastatic prostate cancer patients. CTCs were isolated from the samples using magnetic separation with the cell-surface vimentin(CSV)-specific 84-1 monoclonal antibody that detects epithelial-mesenchymal transitioned (EMT) CTCs. CTCs were enumerated and analyzed for PD-L1 expression using confocal microscopy. PD-L1 expression was detectable in CTCs and was localized in the membrane and/or cytoplasm and nucleus. CTC detection alone was not associated with poor progression-free or overall survival in colorectal cancer or prostate cancer patients, but nuclear PD-L1 (nPD-L1) expression in these patients was significantly associated with short survival durations. These results demonstrated that nPD-L1 has potential as a clinically relevant prognostic biomarker for colorectal and prostate cancer. Our data thus suggested that use of CTC-based models of cancer for risk assessment can improve the standard cancer staging criteria and supported the incorporation of nPD-L1 expression detection in CTCs detection in such models.

Crosstalk between RON and androgen receptor signaling in the development of castration resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.