Lisheng Ge

Lung cancer: a classic example of tumor escape and progression while providing opportunities for immunological intervention.

Lung cancers remain one of the most common and deadly cancers in the world today (12.5% of newly diagnosed cancers) despite current advances in chemo- and radiation therapies. Often, by the time these tumors are diagnosed, they have already metastasized. These tumors demonstrate the classic hallmarks of cancer in that they have advanced defensive strategies allowing them to escape various standard oncological treatments. Immunotherapy is making inroads towards effectively treating other fatal cancers, such as melanoma, glioblastoma multiforme, and castrate-resistant prostate cancers. This paper will cover the escape mechanisms of bronchogenic lung cancer that must be overcome before they can be successfully treated. We also review the history of immunotherapy directed towards lung cancers.

Molecular mechanisms of paraptosis induction: implications for a non-genetically modified tumor vaccine.

Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are “danger signals” that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.

Glioma Cells Display Complex Cell Surface Topographies That Resist the Actions of Cytolytic Effector Lymphocytes

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types—human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing “stem cell-like cells”)—revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor–redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma’s microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-μm transwell chamber’s pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

Big Potassium (BK) ion channels in biology, disease and possible targets for cancer immunotherapy

The Big Potassium (BK) ion channel is commonly known by a variety of names (Maxi-K, KCNMA1, slo, stretch-activated potassium channel, KCa1.1). Each name reflects a different physical property displayed by this single ion channel. This transmembrane channel is found on nearly every cell type of the body and has its own distinctive roles for that tissue type. The BKα channel contains the pore that releases potassium ions from intracellular stores. This ion channel is found on the cell membrane, endoplasmic reticulum, Golgi and mitochondria. Complex splicing pathways produce different isoforms. The BKα channels can be phosphorylated, palmitoylated and myristylated. BK is composed of a homo-tetramer that interacts with β and γ chains. These accessory proteins provide a further modulating effect on the functions of BKα channels. BK channels play important roles in cell division and migration. In this review, we will focus on the biology of the BK channel, especially its role, and its immune response towards cancer. Recent proteomic studies have linked BK channels with various proteins. Some of these interactions offer further insight into the role that BK channels have with cancers, especially with brain tumors. This review shows that BK channels have a complex interplay with intracellular components of cancer cells and still have plenty of secrets to be discovered.

Mitochondrial network in glioma's invadopodia displays an activated state both in situ and in vitro: potential functional implications.

Gliomas are typically characterized by their infiltrative nature, and the prognosis can be linked to the invasive nature of the tumoral cells. Glioblastoma multiforme are very invasive cancers and this contributes to their lethality. The invadopodia are considered essential for their motility. Human glioma cell invadopodia were examined with transmission electron and immunofluorescent microscopy. By electron microscopy, in situ gliomas (fibrillary astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, pilocytic astrocytoma) show mitochondria with a dense matrix condensed configuration, indicating an active state. The mitochondria were frequently in close contact with an extended smooth endoplasmic reticulum displaying an endoplasmic reticulum subfraction associated with mitochondria. Mitochondria were seen within the filopodia that were penetrating into the extracellular matrix. The activated mitochondria and smooth endoplasmic reticulum were also detected within the invadopdia, which was associated microblood vessels. Fluorescent microscopy confirmed that D54 and U251 glioma cells growing in vitro also contained filopodia with mitochondria. The U251 glioma cells’ filopodia that penetrated through 1.2-μm pores of transwell chambers also contained mitocondria, suggesting that the mitochondria are actively involved in the invasion process. Migration and invasion of tumor cells requires an increase in cellular motility and involves formation of lamellipodia, protrusions of the plasma membrane, and individual filopodia [1]. Gliomas are typically characterized by their infiltrative nature, resulting in a poorly demarcated interface between tumor and normal brain tissue. Their poor prognosis can be linked to the invasive nature of these cells. The motility of these tumor cells is correlated with the presence of invadopodia [2], and, consequently, more insight is necessary into their structural and molecular aspects. Evidence of robust invadopodia activity in glioblastoma multiforme cells has been reported [34]. Because of the significant impact of invadopodia in oncological events such as cell invasion and matrix degradation, more insight into structural and molecular aspects is needed [2]. The dynamic assembly of invadopodia is still not well understood [2], and little is known of the alterations in mitochondrial structure and function that contribute to cell mobility [5]. This paper describes two prominent structural features of the mitochondrial network present within the glioma´s invadopodia that we have recently observed. We believe these two features (activated mitochondria and smooth ER, along with mitochondria contained within the filopodia) might provide researchers with possible targets for future therapies that can control glioma invasiveness.

Glioma Big Potassium Channel Expression in Human Cancers and Possible T Cell Epitopes for Their Immunotherapy

Big potassium (BK) ion channels have several spliced variants. One spliced variant initially described within human glioma cells is the glioma BK (gBK) channel. This isoform consists of 34 aa inserted into the intracellular region of the basic BK ion channel. PCR primers specific for this inserted region confirmed that human glioma cell lines and freshly resected surgical tissues from glioblastoma multiforme patients strongly expressed gBK mRNA. Normal human brain tissue very weakly expressed this transcript. An Ab specific for this gBK isoform confirmed that human glioma cells displayed this protein in the cell membrane, mitochondria, Golgi, and endoplasmic reticulum. Within the gBK region, two putative epitopes (gBK1 and gBK2) are predicted to bind to the HLA-A*0201 molecule. HLA-A*0201–restricted human CTLs were generated in vitro using gBK peptide-pulsed dendritic cells. Both gBK1 and gBK2 peptide-specific CTLs killed HLA-A2+/gBK+ gliomas, but they failed to kill non-HLA-A2–expressing but gBK+ target cells in cytolytic assays. T2 cells loaded with exogenous gBK peptides, but not with the influenza M1 control peptide, were only killed by their respective CTLs. The gBK-specific CTLs also killed a variety of other HLA-A*0201+ cancer cells that possess gBK, as well as HLA-A2+ HEK cells transfected with the gBK gene. Of clinical relevance, we found that T cells derived from glioblastoma multiforme patients that were sensitized to the gBK peptide could also kill target cells expressing gBK. This study shows that peptides derived from cancer-associated ion channels maybe useful targets for T cell-mediated immunotherapy.

Immunotherapy of brain cancers: the past, the present, and future directions.

Treatment of brain cancers, especially high grade gliomas (WHO stage III and IV) is slowly making progress, but not as fast as medical researchers and the patients would like. Immunotherapy offers the opportunity to allow the patients own immune system a chance to help eliminate the cancer. Immunotherapys strength is that it efficiently treats relatively small tumors in experimental animal models. For some patients, immunotherapy has worked for them while not showing long-term toxicity. In this paper, we will trace the history of immunotherapy for brain cancers. We will also highlight some of the possible directions that this field may be taking in the immediate future for improving this therapeutic option.

Differential Glioma-Associated Tumor Antigen Expression Profiles of Human Glioma Cells Grown in Hypoxia

Human U251 and D54 glioma cells were tested for expression of 25 glioma-associated tumor antigen precursor proteins (TAPP) under hypoxic (1% O2) or normoxic (21% O2) conditions. Hypoxic glioma cell lines increased their mRNA expression for nine TAPP (Aim2, Art-4, EphA2, EZH2, Fosl1, PTH-rP, Sox 11, Whsc2 and YKL-40), as assessed by quantitative reverse transcriptase real-time/polymerase chain reaction (qRT-PCR). Increased differences with three hypoxic-induced TAPP: EZH2, Whsc2 and YKL-40 were shown at the protein levels by fluorescent antibody staining and quantitative electrophoretic analysis. Two TAPP (MRP3 and Trp1) were down-regulated by hypoxia in glioma cell lines. Growing the glioma cells under hypoxia for 13 days, followed by returning them back to normoxic conditions for 7 days, and restored the original normoxic TAPP profile. Thus, hypoxia was an environmental factor that stimulated the transient expression of these antigens. Intracranial xenografts grown in nude mice derived from U251 cells that had been cultured under neurosphere stem cell conditions showed increased expression of Whsc2 or YKL-40, demonstrating that these in vitro properties of glioma also occur in vivo. Whsc2-specific cytotoxic T lymphocytes killed the hypoxic U251 glioma cells better than normoxic glioma cells. The antigens expressed by hypoxic tumor cells may be a better source of starting tumor material for loading dendritic cells for novel immunotherapy of glioma using tumor-associated antigens.

HCA519/TPX2: a potential T-cell tumor-associated antigen for human hepatocellular carcinoma.

Background Immunotherapy for human hepatocellular cancer (HCC) is slowly making progress towards treating these fatal cancers. The identification of new antigens can improve this approach. We describe a possible new antigen, hepatocellular carcinoma‐associated antigen‐519/targeting protein for Xklp‐2 (HCA519/TPX2), for HCC that might be beneficial for T‐cell specific HCC immunotherapy. Methods HCC was studied for the expression for 15 tumor‐associated antigens considered useful for immunotherapy within three HCC cell lines (HepG2, Hep3B, and PLC/PRF/5), lymphocytes, non‐cancerous livers, and clinical HCC. The expression of tumor antigenic precursor proteins (TAPPs) messenger RNA was first screened by reverse transcriptase quantitative real‐time polymerase chain reaction. Results Four antigens (alpha fetoprotein, aspartyl/asparaginyl βhydroxylase, glypican3 and HCA519/TPX2) proved to be the best expressed TAPPs within the HCC specimens by molecular analyses. HCA519/TPX2 was detected by intracellular cell flow cytometry within HCC cell lines by using a specific antibody towards this TAPP. This antibody also detected the protein within primary HCCs. We synthesized two HCA519/TPX2 peptides (HCA519464–472 and HCA519351–359) which can bind to human leukocyte antigen (HLA)‐A*0201. Dendritic cells pulsed with these peptides stimulated cytolytic T lymphocytes (CTLs). These killer T‐cells lysed HLA‐A*0201+ T2 cells exogenously loaded with the correct specific peptide. The CTLs killed HepG2 (HLA‐A2+ and HCA519+), but not the Hep3B and PLC/PRF/5 cell lines, which are HCA519+ but HLA‐A2‐negative. In silico analysis reveals that HCA519/TPX2 has the inherent ability to bind to a very wide variety of HLA antigens. Conclusion HCA519/TPX2 is a viable immunotarget that should be further investigated within HCC patients.

Using REMBRANDT to Paint in the Details of Glioma Biology: Applications for Future Immunotherapy

Microarray technology developed in the late 1990’s allows one to simultaneously analyze the entire transcriptome of a given population of cells at a single time. It is essentially a microchip with each spot containing about a picogram of DNA immobilized at defined locations. This DNA captures mRNA or cDNA from defined cell populations (tissue, cell line, etc) in a quantitative manner. This technique allows complex patterns of global gene expressions to be quickly and easily detected between two specimens. Microarrays were initially used to describe cell cycle patterns of Saccharomyces cerevisiae [1]. Its powerful potential was quickly recognized and applied towards cancer. The first successful application of this technology was to show that diffuse large cell B lymphomas had two different subtypes. One subset of this cancer was easily treatable with chemotherapy; whereas, the second phenotype required a more aggressive therapy [2].