Hyperthermic intraperitoneal chemotherapy enhances antitumor effects on ovarian cancer through immune-mediated cancer stem cell targeting.

PURPOSE: We aimed to determine the effects and possible mechanisms of hyperthermic intraperitoneal chemotherapy (HIPEC) in targeting ovarian cancer stem-like cells (CSCs). METHODS: Murine ovarian cancer cell lines presenting CSC surface markers were grown intraperitoneally in both immunocompetent and immunodeficient mice, which were then treated by intraperitoneal hyperthermia with the chemotherapeutic agents: paclitaxel and cisplatin. Tumor growth was measured by non-invasive luminescent imaging. Intraperitoneal immune cells, such as CD4+, CD8+ T cells, macrophages, and dendritic cells, were evaluated through flow cytometry analysis. RESULTS: Combined hyperthermia and chemotherapy exhibited an efficient therapeutic effect in the immunocompetent mice. However, a similar effect was not observed in the immunodeficient mice. Intraperitoneal hyperthermia increased the number of Intraperitoneal macrophages and dendritic cells that were lost due to chemotherapy. Compared with ovarian cancer bulk cells, CSCs were more susceptible to phagocytosis by macrophages. CONCLUSION: We demonstrated that the superior therapeutic efficacy and reduced proportion of CSCs associated with intraperitoneal hyperthermic chemotherapy were immune-related. Hyperthermia recruits the phagocytes that target surviving CSCs after chemotherapy. These results provide a novel mechanism for the efficacy of HIPEC in treating ovarian cancer.

Effect of hyperthermia on improving neutrophil restoration after intraperitoneal chemotherapy.

Purpose: Intraperitoneal (IP) chemotherapy has several benefits but also can have severe hematologic side effects. We compared the effects of hyperthermic intraperitoneal chemotherapy (HIPEC) and conventional IP chemotherapy on bone marrow suppression and evaluated whether HIPEC increased neutrophil recovery.Methods: HIPEC or IP chemotherapy was administered to ovarian cancer-bearing mice. Bone marrow progenitor cell colony-forming unit (CFU) count, serum cytokine levels, and peripheral leukocyte count after HIPEC and IP chemotherapy were compared.Results: Peripheral neutrophil count, cytokine (G-CSF and CXCL1/KC) levels, and bone marrow progenitor cell CFU count were significantly higher after HIPEC than after IP chemotherapy.Conclusions: Hyperthermia increased the serum neutrophil-recruiting cytokine levels and reduced the magnitude of chemotherapy-induced neutropenia. Thus, HIPEC improved neutrophil and bone marrow recovery compared with conventional IP chemotherapy.

Immune mechanism of the antitumor effects generated by bortezomib.

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8+ T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8+ T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8+ T cells. CD11c+ DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c+ DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.

Immune vulnerability of ovarian cancer stem-like cells due to low CD47 expression is protected by surrounding bulk tumor cells.

Recurrence of advanced epithelial ovarian cancer is common despite optimal surgical debulking and initial favorable responses to chemotherapy. Evidences suggest that cancer stem cells (CSCs) have inherent resistance to conventional therapies such as chemotherapy and play a decisive role in cancer recurrence. Cancer stem cells are also believed to be able to evade immunological attack. However, this study showed a different scenario in which cancer stem-like cells are more vulnerable to immunosurveillance. Our study demonstrated that isolated murine cancer stem-like cells, stem cell antigen (SCA)-1+ ID8 and CD133+ HM-1 cells, were susceptible to phagocytosis by macrophages and consequent CD8+ T cell immunity. The increased phagocytosis of these stem cell-like cells is attributed to low CD47 protein expression. SCA-1+ ID8 cells were able to grow in syngeneic mice but were soon rejected. Restoring CD47 expression delayed this immune-mediated rejection. SCA-1+ ID8 cells showed rapid growth by mixing with bulk ID8 cells. These results suggest that stem-like cells could be protected by surrounding non-stem cancer cells from immune attack. Similarly, both isolated human CD24-/low SKOV3 stem-like cells and spheroid OVCAR3 cells expressed lower CD47 levels. Our study provided novel insights into the immune characteristics of CSCs within a tumor microenvironment. The results might lead to the design of more effective treatment strategies for ovarian cancer.

Clinical significance of c-Met and phospho-c-Met (Tyr1234/1235) in ovarian cancer.

OBJECTIVE: c-Met is expressed in human ovarian cancer tissues, and its phosphorylation activates signaling cascades that might affect the behavior of cancer cells. In this study, we evaluated the association of c-Met and phosphorylated c-Met (phospho-c-Met) expressions with the clinical outcomes of ovarian cancer patients. MATERIALS AND METHODS: Archived tissue from surgical specimens of 269 ovarian cancer patients who underwent a debulking operation in MacKay Memorial Hospital between 2004 and 2012 were collected. Tissue microarrays were stained with anti-Met and anti-phospho-Met (Tyr1234/1235) monoclonal antibodies. Immunostaining intensity was scored on a scale of 0-3+. High expression was defined as more than 50% of moderate and intense staining. Patients' clinical data were reviewed until April 2017 for analysis. RESULTS: The proportion of high c-Met expression was significantly higher in patients with cancer in early stages (Federation of Gynecology and Obstetrics stages I and II) and low histologic grades (grades 1 and 2) (79.70%, p = 0.0008 and 80.15%, p ≤ 0.0001, respectively). However, no association was found between phospho-c-Met and FIGO stage or the histologic grade. Ovarian clear cell carcinoma and mucinous carcinoma had much higher c-Met expression (95.16% and 87.10%, p ≤ 0.0001 and p = 0.0292, respectively). Although the overall survival did not differ significantly, low expressions of c-Met and phospho-c-Met were obviously associated with poor progression-free survival respectively (p = 0.0034, HR: 0.5264, 95% CI: 0.3326-0.8330 and p = 0.0136, HR: 0.5626, 95% CI: 0.3709-0.8535). CONCLUSION: Low c-Met expression was associated with poor clinical outcomes.

Antitumor effects of BMS-777607 on ovarian cancer cells with constitutively activated c-MET.

OBJECTIVE: Tyrosine-protein kinase MET (c-MET) has been reported to be a prognostic marker and suitable therapeutic target for ovarian cancer. BMS-777607, a small molecule, can inhibit MET and other protein kinase activities. The present study was conducted to investigate the mechanism of action and antitumor effect of BMS-777607 on ovarian cancer cells with constitutively activated c-MET. MATERIALS AND METHODS: Ovarian cancer cells with constitutively activated c-MET were first identified through Western blot analysis. Bio-behaviors, including signal transduction, proliferation, apoptosis, and migration, of the cells with constitutively activated c-MET were evaluated after BMS-777607 treatment. Liu's stain and immunological staining of α-tubuline were performed to evaluate the ploidy of the cells. A xenograft mouse model was also used to evaluate the antitumor effects of BMS-777607 on ovarian cancer cells with constitutively activated c-MET. RESULTS: BMS-777607 could induce the highest inhibition of cell growth in ovarian cancer cells constitutively expressing c-MET. Treating SKOV3 cells with BMS-777607 could reduce c-MET activation and inhibit downstream cell signaling, thus causing cell apoptosis and polyploidy as well as cell cycle and cell migration inhibition. This molecule also inhibited tumor growth in a mouse xenograft model of SKOV3 ovarian cancer cells in vivo. CONCLUSION: BMS-777607 exhibits antitumor effects on ovarian cancer cells that constitutively express c-MET through c-MET signaling blockade and the inhibition of Aurora B activity. Combination treatments to enhance the effects of BMS-777607 warrant investigation in the future.

Less circulating mucosal-associated invariant T cells in patients with cervical cancer.

OBJECTIVE: Mucosal-associated invariant T cells (MAITs) are important for immune defense against infectious pathogens and regulation of various inflammatory diseases. However, their roles in cancer are rarely reported. Since cervical cancer is one of the diseases involving mucosal tissue, we try to investigate the association between circulating MAITs and cervical cancer. MATERIALS AND METHODS: Blood samples were obtained from patients with cervical cancer (n = 47) and healthy individuals (n = 39). We determined phenotypic MAITs in peripheral blood mononuclear cells (PBMCs) and evaluated the percentage of MAITs in CD3+ cells by flow cytometry. The percentage of MAITs was stratified according to Federation of Gynecology and Obstetrics (FIGO) staging system in patients with cervical cancer. Progression-free survival (PFS) with respect to the amount of MAITs was also analyzed. RESULTS: The percentage of circulating MAITs in patients with cervical cancer was significantly lower than in healthy group (0.987% vs. 4.008%, p < 0.0001). In subgroup analysis, though not statistically significant, it showed a trend of lower percentage of circulating MAITs in cervical cancer patients with FIGO stage II-IV disease than in patients with FIGO stage I disease (0.4045% vs. 1.098%, p = 0.11). A trend of poor PFS in patients with lower circulating MAITs was also noted. CONCLUSION: MAITs play a crucial role in cancer immunity. The decrease of MAITs in peripheral blood is related to cervical cancer. There is a trend of lower percentage of MAITs in advanced stages and lower percentage of MAITs towards poor PFS in patients with cervical cancer.

Intestinal parasitic infections: Current prevalence and risk factors among schoolchildren in capital area of the Republic of Marshall Islands.

Intestinal parasitic infections (IPIs) among schoolchildren in Republic of Marshall Islands (RMI) largely remains unknown, thus investigation on IPIs status to establish the baseline data is urgently needed. This cross-sectional study intended to investigate the current IPIs status and associated risk factors among schoolchildren at capital of RMI. Single stool sample from 400 schoolchildren (207 boys and 193 girls) aged 9.73±2.50 yrs old was examined by employing merthiolate-iodine-formaldehyde concentration method. Demographic characteristics, uncomfortable symptoms and risk factors were obtained by questionnaires investigation. The overall prevalence of IPIs in schoolchildren was 22.8% (91/400), of them 24.2% harbored at least 2 different parasites. Notably, the majority was infected by waterborne protozoan parasites (82.4%, 75/91). Nine different intestinal parasites have been identified, of which six were pathogenic including Hook worm, Trichuris trichiura, Enterobius vermicularis, Entamoeba histolytica/dispar, Giardia intestinalis and Blastocystis hominis. Schoolchildren who ever complained dizziness or headache showed a significant higher prevalence of pathogenic IPIs than those who did not (p<0.05). Schoolchildren who lived in urban area than rural area had higher chance to acquire pathogenic IPIs (p=0.03). However, none of risk factors were identified to be associated with pathogenic IPIs.

Nanoparticle-induced intraperitoneal hyperthermia and targeted photoablation in treating ovarian cancer.

Hyperthermic intraperitoneal chemotherapy is effective in treating various intra-abdominal malignancies. However, this therapeutic modality can only be performed during surgical operations and cannot be used repeatedly. We propose repeatedly noninvasive hyperthermia mediated by pegylated silica-core gold nanoshells (pSGNs) in vivo with external near-infrared (NIR) laser irradiation. This study demonstrated that repeated photothermal treatment can effectively eliminate intraperitoneal tumors in mouse ovarian cancer models without damage of normal tissues. By conjugating pSGNs with anti-human CD47 monoclonal antibody, a significant photoablative effect can be achieved using lower amount of pSGNs and shorter NIR laser irradiation. Conjugated pSGNs specifically targeted and bound to cancer cells inside the peritoneal cavity. Our results indicate the possibility of a noninvasive method of repeated hyperthermia and photoablative therapies using nanoparticles. This has substantial clinical potential in treating ovarian and other intraperitoneal cancers.

Dose-dense chemotherapy improves mechanisms of antitumor immune response.

Dose-dense (DD) regimens of combination chemotherapy may produce superior clinical outcomes, but the basis for these effects are not completely clear. In this study, we assessed whether a DD combinatorial regimen of low-dose cisplatin and paclitaxel produces superior immune-mediated efficacy when compared with a maximum tolerated dose (MTD) regimen in treating platinum-resistant ovarian cancer as modeled in mice. Immune responses generated by the DD regimen were identified with regard to the immune cell subset responsible for the antitumor effects observed. The DD regimen was less toxic to the immune system, reduced immunosuppression by the tumor microenvironment, and triggered recruitment of macrophages and tumor-specific CD8(+) T-cell responses to tumors [as determined by interleukin (IL)-2 and IFN-γ secretion]. In this model, we found that the DD regimen exerted greater therapeutic effects than the MTD regimen, justifying its further clinical investigation. Fourteen patients with platinum-resistant relapse of ovarian cancer received DD chemotherapy consisting of weekly carboplatin (AUC2) and paclitaxel (60-80 mg/m(2)) as the third- or fourth-line treatment. Serum was collected over the course of treatment, and serial IFN-γ and IL-2 levels were used to determine CD8(+) T-cell activation. Of the four patients with disease control, three had serum levels of IL-2 and IFN-γ associated with cytotoxic CD8(+) T-cell activity. The therapeutic effect of the DD chemotherapy relied on the preservation of the immune system and the treatment-mediated promotion of tumor-specific immunity, especially the antitumor CD8(+) T-cell response. Because the DD regimen controlled drug-resistant disease through a novel immune mechanism, it may offer a fine strategy for salvage treatment.

Intra-peritoneal hyperthermia combining α-galactosylceramide in the treatment of ovarian cancer.

The purpose of this study was to investigate the anti-tumor effect and potential mechanisms of i.p. hyperthermia in combination with α-galactosylceramide (α-GalCer) for the treatment of ovarian cancer. In this study, immuno-competent tumor models were established using murine ovarian cancer cell lines and treated with i.p. hyperthermia combining α-GalCer. Th1/Th2 cytokine expression profiles in the serum, NK cell cytotoxicity and phagocytic activities of dendritic cells (DCs) were assayed. We also analyzed the number of CD8(+)/IFN-γ(+) tumor specific cytotoxic T cells, as well as the tumor growth based on depletion of lymphocyte sub-population. Therapeutic effect on those ovarian tumors was monitored by a non-invasive luminescent imaging system. Intra-peritoneal hyperthermia induced significant pro-inflammatory cytokines expression, and sustained the response of NK and DCs induced by α-GalCer treatment. The combination treatment enhanced the cytotoxic T lymphocyte (CTL) immune response in two mouse ovarian cancer models. This novel treatment modality by combination of hyperthermia and glycolipid provides a pronounced anti-tumor immune response and better survival. In conclusion, intra-peritoneal hyperthermia enhanced the pro-inflammatory cytokine secretion and phagocytic activity of DCs stimulated by α-GalCer. The subsequent CTL immune response induced by α-GalCer was further strengthened by combining with i.p. hyperthermia. Both innate and adaptive immunities were involved and resulted in a superior therapeutic effect in treating the ovarian cancer.

Silencing of astrin induces the p53-dependent apoptosis by suppression of HPV18 E6 expression and sensitizes cells to paclitaxel treatment in HeLa cells.

Astrin is a microtubule-associated protein and localizes with mitotic spindles in the M-phase. We silenced the expression of astrin protein and tested the cell viability in response to paclitaxel treatment in paclitaxel-sensitive and paclitaxel-resistant cells. We found that the absence of astrin by siRNA resulted in the activation of a p53-dependent apoptosis, which elevated pro-apoptotic Bax expression and increased the activity of caspase-3 in astrin-depleted cells. The HPV18 E6 transcription was found to be inhibited along with the increase expression of p53. Intriguingly, the expression of astrin decreased in paclitaxel-sensitive HeLa cells but remained steady in paclitaxel-resistant cells in response to paclitaxel treatment. Furthermore, we identified that the depletion of astrin caused more cell death both in paclitaxel-sensitive and -resistant cells in combination with paclitaxel treatment. These findings suggest that the silencing of astrin induce a p53-dependent apoptosis and has an additive effect on paclitaxel treatment.

Antibody detection of SARS-CoV spike and nucleocapsid protein.

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.