Yu-Shan Wang

Combination of conformal radiotherapy and intratumoral injection of adoptive dendritic cell immunotherapy in refractory hepatoma.

A phase 1 study was conducted to assess the safety and immunologic response induced by direct injection of autologous immature dendritic cells (DCs) into tumor under radiotherapy in advanced hepatoma patients. Patients with advanced/metastatic stage hepatoma not suitable for surgery or transarterial embolization were enrolled. Groups of patients received two vaccinations. Each vaccination consisted of intratumoral injections of autologous immature DCs in four dose cohorts of 5 × 106, 1.5 × 107, 3 × 107, and 5 × 107 cells 2 days after a single fraction of conformal radiotherapy of 8 Gy. The second vaccination was performed 3 weeks later. Of the 14 patients entered, 12 completed two cycles of vaccination. The treatment was well tolerated at any of the dose levels. Six patients had mild transient fever (grade 1-2) with chill reactions, three patients developed grade 1 fatigue, and one patient developed mild myalgia and arthralgia after DC injections. There was no evidence of clinically manifested autoimmune disease. There were two partial responses and four minor responses. A decrease in the alpha-fetoprotein (AFP) level of more than 50% was found in three patients. Ten patients had completed immunologic response evaluation 2 weeks after the second cycle of vaccination. The AFP-specific immune response was evident in eight patients examined by cytokine release assay and in seven patients by ELISPOT assay. Six patients showed an increased NK cell cytotoxic activity after vaccination. These data suggest that the combination of intratumoral injection of DCs and conformal radiotherapy is safe and can induce tumor-specific and innate immunity.

Apolipoprotein C1 (APOC1) as a novel diagnostic and prognostic biomarker for lung cancer: A marker phase I trial

Tumor cells continuously evolve over time in response to host pressures. However, explanations as to how tumor cells are influenced by the inflammatory tumor microenvironment over time are, to date, poorly defined. We hypothesized that prognostic biomarkers could be obtained by exploring the expression of inflammation‐associated genes between early and late stage lung cancer tumor samples.

Simultaneous activation and inhibition of autophagy sensitizes cancer cells to chemotherapy.

While combined chemotherapy (CT) with an autophagy inducer and an autophagy inhibitor appears paradoxical, it may provide a more effective perturbation of autophagy pathways. We used two dissimilar cell lines to test the hypothesis that autophagy is the common denominator of cell fate after CT. HA22T cells are characterized by CT-induced apoptosis and use autophagy to prevent cell death, while Huh7.5.1 cells exhibit sustained autophagic morphology after CT. Combined CT and rapamycin treatment resulted in a better combination index (CI) in Huh7.5.1 cells than combined CT and chloroquine, while the reverse was true in HA22T cells. The combination of 3 drugs (triplet drug treatment) had the best CI. After triplet drug treatment, HA22T cells switched from protective autophagy to mitochondrial membrane permeabilization and endoplasmic reticulum stress response-induced apoptosis, while Huh7.5.1 cells intensified autophagic lethality. Most importantly, both cell lines showed activation of Akt after CT, while the triplet combination blocked Akt activation through inhibition of phospholipid lipase D activity. This novel finding warrants further investigation as a broad chemosensitization strategy.

Reciprocal Complementation of the Tumoricidal Effects of Radiation and Natural Killer Cells

The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.

Improving Radioresponse Through Modification of the Tumor Immunological Microenvironment

Radioresponse is influenced by factors apart from the targeted cancer cells; in fact, endothelial cells and infiltrating immune cells within the tumor microenvironment (TME) are the two main components affecting the outcome of radiotherapy. The benefits of fractionated radiotherapy are attenuated through the upregulation of hypoxia inducible factor-1 α and vascular endothelial growth factor. The therapeutic effect of antiangiogenic agents is counteracted by the mobilization of endogenous proangiogenic cells to the TME. This study highlights the importance of radiation timing within a vascular normalization window and discusses the importance of immune cells that comprise the microenvironment. A balance between favorable tumor-infiltrating immune cells, including cytotoxic T cells, natural killer cells, and dendritic cells, and the unfavorable cells, such as tumor-associated macrophages and regulatory T cells, determines the final tumor-control probability. The reciprocal complementation between combinations of radiotherapy and immunotherapy strategies through modulation of the tumor immunological microenvironment may yield promising results in the future.

Improving Detection Accuracy of Lung Cancer Serum Proteomic Profiling via Two-Stage Training Process

BackgroundSurface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) is a frequently used technique for cancer biomarker research. The specificity of biomarkers detected by SELDI can be influenced by concomitant inflammation. This study aimed to increase detection accuracy using a two-stage analysis process.MethodsSera from 118 lung cancer patients, 72 healthy individuals, and 31 patients with inflammatory disease were randomly divided into training and testing groups by 3:2 ratio. In the training group, the traditional method of using SELDI profile analysis to directly distinguish lung cancer patients from sera was used. The two-stage analysis of distinguishing the healthy people and non-healthy patients (1st-stage) and then differentiating cancer patients from inflammatory disease patients (2nd-stage) to minimize the influence of inflammation was validated in the test group.ResultsIn the test group, the one-stage method had 87.2% sensitivity, 37.5% specificity, and 64.4% accuracy. The two-stage method had lower sensitivity (> 70.1%) but statistically higher specificity (80%) and accuracy (74.7%). The predominantly expressed protein peak at 11480 Da was the primary splitter regardless of one- or two-stage analysis. This peak was suspected to be SAA (Serum Amyloid A) due to the similar m/z countered around this area. This hypothesis was further tested using an SAA ELISA assay.ConclusionsInflammatory disease can severely interfere with the detection accuracy of SELDI profiles for lung cancer. Using a two-stage training process will improve the specificity and accuracy of detecting lung cancer.

Serum amyloid a as a predictive marker for radiation pneumonitis in lung cancer patients.

PURPOSE To investigate serum markers associated with radiation pneumonitis (RP) grade ≥3 in patients with lung cancer who were treated with radiation therapy. METHODS AND MATERIALS Pretreatment serum samples from patients with stage Ib-IV lung cancer who developed RP within 1 year after radiation therapy were analyzed to identify a proteome marker able to stratify patients prone to develop severe RP by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Dosimetric parameters and 3 biological factors were compared. RESULTS Serum samples from 16 patients (28%) with severe RP (grade 3-4) and 42 patients (72%) with no or mild RP (grade 0-2) were collected for analysis. All patients received a median of 54 Gy (range, 42-70 Gy) of three-dimensional conformal radiation therapy with a mean lung dose (MLD) of 1502 cGy (range, 700-2794 cGy). An m/z peak of 11,480 Da was identified by SELDI-TOF-MS, and serum amyloid A (SAA) was the primary splitter serum marker. The receiver operating characteristic area under the curve of SAA (0.94; 95% confidence interval [CI], 0.87-1.00) was higher than those of C-reactive protein (0.83; 95% CI, 0.72-0.94), interleukin-6 (0.79; 95% CI, 0.65-0.94), and MLD (0.57; 95% CI, 0.37-0.77). The best sensitivity and specificity of combined SAA and MLD for predicting RP were 88.9% and 96.0%, respectively. CONCLUSIONS Baseline SAA could be used as an auxiliary marker for predicting severe RP. Extreme care should be taken to limit the lung irradiation dose in patients with high SAA.

Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy.

Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C′-PSA or rHsp70C′-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

In vitro comparison of conventional hyperthermia and modulated electro-hyperthermia.

Radiofrequency-induced hyperthermia (HT) treatments for cancer include conventional capacitive coupling hyperthermia (cCHT) and modulated electro-hyperthermia (mEHT). In this study, we directly compared these methods with regard to in vitro cytotoxicity and mechanisms of action under isothermal conditions. Hepatoma (HepG2) cells were exposed to HT treatment (42°C for 30 min) using mEHT, cCHT or a water bath. mEHT produced a much higher apoptosis rate (43.1% ± 5.8%) than cCHT (10.0% ± 0.6%), the water bath (8.4% ± 1.7%) or a 37°C control (6.6% ± 1.1%). The apoptosis-inducing effect of mEHT at 42°C was similar to that achieved with a water bath at 46°C. mEHT also increased expression of caspase-3, 8 and 9. All three hyperthermia methods increased intracellular heat shock protein 70 (Hsp70) levels, but only mEHT greatly increased the release of Hsp70 from cells. Calreticulin and E-cadherin levels in the cell membrane also increased after mEHT treatment, but not after cCHT or water bath. These results suggest that mEHT selectively deposits energy on the cell membrane and may be a useful treatment modality that targets cancer cell membranes.