Izhar Singh Batth

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.

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.

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.

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.

Palmatine inhibits growth and invasion in prostate cancer cell

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.

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.

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

ABSTRACT 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.

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

ABSTRACT 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.

Abstract 1931: The first circulating tumor cell detection technique from frozen PBMCs

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 application is not fully investigated, partly due to the necessity for fresh blood samples and the lack of CTC specific antibody. Of note, there is no CTC detection tool for pediatric tumors. To overcome these drawbacks, we developed a protocol for CTC capture from frozen peripheral blood mononuclear cells (PBMCs) based on the cell-surface vimentin (CSV) antibody 84-1. CSV is a unique marker compared to others such as EpCAM because it is only specific to tumor cells and has been demonstrated to be a viable target for isolation and enumeration of CTCs across tumor types. In this study, we report the first CTC isolation technology from frozen PBMCs of osteosarcoma patients using the CSV antibody 84-1. The CTCs captured using this new protocol were validated by single cell gene sequencing analysis and mesenchymal marker α-SMA staining. Moreover, spiking analysis was also performed to ensure the specificity and sensitivity of this unique CTC capture analysis. Linear regression analysis yielded a positive correlation between the number of detected sarcoma cells and the number of sarcoma cells spiked in both fresh and frozen PBMCs. In summary, our results demonstrate for the first time, a technology to specifically detect and isolate mesenchymal origin CTCs from frozen PBMCs. This technology can be easily expanded to other types of cancers including pediatric tumors because the antibody used is universal and CTC specific. Such a technology will boost the feasibility and utility of CTC-based diagnosis and therapeutic treatment monitoring in a centralized laboratory. Citation Format: Heming Li, Izhar Singh Batth, Xueqing Xia, Frank J. Hsu, Neeta Somaiah, Keila Enitt Torres, Ruoyu Wang, Shulin Li. The first circulating tumor cell detection technique from frozen PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1931. doi:10.1158/1538-7445.AM2017-1931

Abstract 1941: Cell surface vimentin is a novel marker for CTC detection in neuroblastoma

Among children in the United States, brain cancers now account for most number of cancer-related deaths. Neuroblastomas (NB) and sarcomas account for 13% of all childhood cancers in the United States. Despite significant progress yielding increased 5-year survival, 1250 deaths are expected this year from childhood cancers and cancer incidence has been steadily increasing. One of the key contributors to cancer mortality is tumor metastasis. It is generally believed that CTCs are shed into the circulation from primary tumors and contain unique driver mutations enabling their aggressive phenotype. However, detection of CTCs from NB is difficult as there is no direct method. Our lab has developed a monoclonal antibody targeting vimentin on the cells’ surface. Cell surface vimentin (CSV) is only observed in tumor cells; it remains intracellular in normal cells. We have published data demonstrating the superior specificity and sensitivity of our approach in breast, colon, and prostate cancer. Here, we report direct CTC detection in NB using our CSV mAb. As part of our collaborative effort analyzing NB blood samples from a multicenter Phase II trial, we present novel discoveries regarding treatment of NB with difluoromethylornithine (DFMO) and its effects on CTC release into the circulation in patients under remission. Among our observations, approximately ~1/3 patients have low or no CTCs while ~1/4 with high numbers of CTC are responding to DFMO treatment. Remaining patients show oscillating CTC numbers. Further, treatment with DFMO showed a statistically significant decline in CTCs after the beginning of therapy. Ongoing work including whole genome analysis can possibly reveal new insight into CTCs’ entering the circulation. These data demonstrate that our CTC capture using CSV is a novel and unique approach for detection in NB. Citation Format: Izhar S. Batth, Heming Li, Giselle Saulnier Sholler, Shulin Li. Cell surface vimentin is a novel marker for CTC detection in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2017-1941