Ultrasound Doppler as an Imaging Modality for Selection of Murine 4T1 Breast Tumors for Combination Radiofrequency Hyperthermia and Chemotherapy

Noninvasive radiofrequency-induced (RF) hyperthermia has been shown to increase the perfusion of chemotherapeutics and nanomaterials through cancer tissue in ectopic and orthotopic murine tumor models. Additionally, mild hyperthermia (37°C-45°C) has previously shown a synergistic anticancer effect when used with standard-of-care chemotherapeutics such as gemcitabine and Abraxane. However, RF hyperthermia treatment schedules remain unoptimized, and the mechanisms of action of hyperthermia and how they change when treating various tumor phenotypes are poorly understood. Therefore, pretreatment screening of tumor phenotypes to identify key tumors that are predicted to respond more favorably to hyperthermia will provide useful mechanistic data and may improve therapeutic outcomes. Herein, we identify key biophysical tumor characteristics in order to predict the outcome of combinational RF and chemotherapy treatment. We demonstrate that ultrasound imaging using Doppler mode can be utilized to predict the response of combinational RF and chemotherapeutic therapy in a murine 4T1 breast cancer model.     

Evaluation of poly-mechanistic antiangiogenic combinations to enhance cytotoxic therapy response in pancreatic cancer

Gemcitabine (Gem) has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). The present study investigated combinations of gemcitabine with antiangiogenic agents of various mechanisms for PDAC, including bevacizumab (Bev), sunitinib (Su) and EMAP II. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo experiments were performed via murine xenografts. Inhibition of in vitro proliferation of AsPC-1 PDAC cells by gemcitabine (10 μM), bevacizumab (1 mg/ml), sunitinib (10 μM) and EMAP (10 μM) was 35, 22, 81 and 6 percent; combination of gemcitabine with bevacizumab, sunitinib or EMAP had no additive effects. In endothelial HUVECs, gemcitabine, bevacizumab, sunitinib and EMAP caused 70, 41, 86 and 67 percent inhibition, while combination of gemcitabine with bevacizumab, sunitinib or EMAP had additive effects. In WI-38 fibroblasts, gemcitabine, bevacizumab, sunitinib and EMAP caused 79, 58, 80 and 29 percent inhibition, with additive effects in combination as well. Net in vivo tumor growth inhibition in gemcitabine, bevacizumab, sunitinib and EMAP monotherapy was 43, 38, 94 and 46 percent; dual combinations of Gem+Bev, Gem+Su and Gem+EMAP led to 69, 99 and 64 percent inhibition. Combinations of more than one antiangiogenic agent with gemcitabine were generally more effective but not superior to Gem+Su. Intratumoral proliferation, apoptosis and microvessel density findings correlated with tumor growth inhibition data. Median animal survival was increased by gemcitabine (26 days) but not by bevacizumab, sunitinib or EMAP monotherapy compared to controls (19 days). Gemcitabine combinations with bevacizumab, sunitinib or EMAP improved survival to similar extent (36 or 37 days). Combinations of gemcitabine with Bev+EMAP (43 days) or with Bev+Su+EMAP (46 days) led to the maximum survival benefit observed. Combination of antiangiogenic agents improves gemcitabine response, with sunitinib inducing the strongest effect. These findings demonstrate advantages of combining multi-targeting agents with standard gemcitabine therapy for PDAC.     

Laser hyperthermia of tumors using gold nanoparticles monitored by optical coherence tomography and acoustic thermometry

Local laser hyperthermia of grafted RShM-5 tumors in mice with the use of plasmon resonant gold nanoparticles has been carried out. Accumulation of particles in the tumor was monitored in vivo noninvasively by optical coherence tomography. Thereby it was determined that the maximal content of nanoparticles in the tumor was reached within 5 h after intravenous administration, and laser hyperthermia was performed at this time. Monitoring the tumor temperature during the treatment by IR thermography and acoustic thermometry showed that the nanoparticles provided efficient temperature elevation inside the tumor as well as more selective heating. Local laser hyperthermia with gold nanoparticles, but not the laser exposure alone, substantially inhibited tumor growth in several days after a single session.     

A PAUF-neutralizing antibody targets both carcinoma and endothelial cells to impede pancreatic tumor progression and metastasis

Pancreatic adenocarcinoma up-regulated factor (PAUF) is expressed in pancreatic ductal adenocarcinoma (PDAC) and plays an important role in tumor progression and metastasis. Here we evaluate the anti-tumor efficacy of a human monoclonal antibody against PAUF, PMAb83, to provide a therapeutic intervention to treat the disease. PMAb83 reduced tumor growth and distant metastasis in orthotopically xenografted mice of human PDAC cells. PMAb83 treatment retarded proliferation along with weakened aggressiveness traits of the carcinoma cells. AKT/β-catenin signaling played a role in the carcinoma cell proliferation and the treated xenograft tumors exhibited reduced levels of β-catenin and cyclin D1. Moreover PMAb83 abrogated the PAUF-induced angiogenic responses of endothelial cells, reducing the density of CD31⁺ vessels in the treated tumors. In combination with gemcitabine, PMAb83 conferred enhanced survival of xenografted mice by about twofold compared to gemcitabine alone. Taken together, our findings show that PMAb83 treatment decreases the aggressiveness of carcinoma cells and suppresses tumor vascularization, which culminates in mitigated tumor growth and metastasis with improved survival in PDAC mouse models.     

Electromagnetic treatment to old Alzheimer's mice reverses β-amyloid deposition, modifies cerebral blood flow, and provides selected cognitive benefit

Few studies have investigated physiologic and cognitive effects of "long-term" electromagnetic field (EMF) exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25-1.05 W/kg) by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer's transgenic (Tg) mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain β-amyloid (Aβ) aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21-27 month) Tg mice over a 2-month period reverses their very advanced brain Aβ aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature) during EMF "ON" periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated Aβ (Tg mice) and slight body hyperthermia during "ON" periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer's Tg mice and normal mice, as well as reversal of advanced Aβ neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF treatment against AD.     

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.     

Targeted therapies in pancreatic cancer: Promises and failures

Pancreatic ductal adenocarcinoma (PDAC) is an incidence rate nearly equal to its mortality rate. The poor prognosis of the disease can be explained by the absence of effective biomarkers for screening and early detection, together with the aggressive behavior and resistance to the currently available chemotherapy. The therapeutic failure can also be attributed to the inter-/intratumor genetic heterogeneity and the abundance of tumor stroma that occupies the majority of the tumor mass. Gemcitabine is used in the treatment of PDAC; however, the response rate is less than 12%. A recent phase III trial revealed that the combination of oxaliplatin, irinotecan, fluorouracil, and leucovorin could be an option for the treatment of metastatic PDAC patients with good performance status, although these approaches can result in high toxicity level. Further investigations are required to develop innovative anticancer agents that either improve gemcitabine activity, within novel combinatorial approaches or acts with a better efficacy than gemcitabine. The aim of the current review is to give an overview of preclinical and clinical studies targeting key dysregulated signaling pathways in PDAC.     

The efficacy of a novel, dual PI3K/mTOR inhibitor NVP-BEZ235 to enhance chemotherapy and antiangiogenic response in pancreatic cancer

Gemcitabine has limited clinical benefits for pancreatic ductal adenocarcinoma (PDAC). The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in PDAC. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival experiments were performed in murine xenografts. BEZ235 caused a decrease in phospho-AKT and phospho-mTOR expression in PDAC (AsPC-1), endothelial (HUVECs), and fibroblast (WI-38) cells. BEZ235 inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on proliferation inhibition were observed in the BEZ235-gemcitabine combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with gemcitabine and EMAP, induced apoptosis in AsPC-1, HUVECs, and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and gemcitabine monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235 + gemcitabine (30 days, P = 0.007), BEZ235 + EMAP (27 days, P = 0.02), gemcitabine + EMAP (31 days, P = 0.001), and BEZ235 + gemcitabine + EMAP (33 days, P = 0.004). BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of gemcitabine and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment.     

Inhibition of Lewis lung carcinoma in mice by local microwave hyperthermia combined with immunomodulating Propionibacterium granulosum KP-45

C-57 BL/6 mice with Lewis lung carcinoma were treated 2 weeks after tumor implantation with local microwave hyperthermia (2450 MHz, tumor temperature 43.5 degrees C, 30 min) and/or intratumoral or intraperitoneal injection of 1 mg cell walls of Propionibacterium granulosum KP-45. Tumor growth, survival time of the animals, and the delayed skin hypersensitivity to oxazolone were followed up, as well as the 3H-thymidine uptake of tumorous tissues and the number of lung metastases. The combined treatment of microwave hyperthermia with immunomodulating P. granulosum KP-45 resulted in significantly stronger inhibition of tumor growth than with each of these methods alone. The number of lung metastases could be significantly lowered, and the skin reactivity to oxazolone remained enhanced during the whole observation period (over 70 days after tumor implantation). The implications of the test observation are discussed.     

Tumor Selective Hyperthermia Induced by Short-Wave Capacitively-Coupled RF Electric-Fields

There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.     

Effects of low level microwave radiation on carcinogenesis in Swiss Albino mice

This study concerns with the multiple treatment of the target site to potent carcinogen and the super imposition of low level radiofrequency and microwave radiation. Swiss albino mice (male) were used for this investigation. The study has been divided in two parts, part A: a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) 100 μg/animal was applied topically on the skin of mice and were exposed to 112 MHz amplitude modulated (AM) at 16 Hz (power density 1.0 mW/cm(2), specific absorption rate (SAR) 0.75 W/kg). Similarly after a single dose of DMBA, mice were exposed to 2.45 GHz radiation (power density of 0.34 mW/cm(2), SAR, 0.1 W/kg), 2 h/day, 3 days a week for a period of 16 weeks. The two sets of experiments were carried out separately. Part B: mice were transplanted intraperitoneally (ip) with ascites 8 × 10(8) (Ehrlich-Lettre ascites, strain E) carcinoma cells per mouse. These mice were exposed to 112 MHz amplitude modulated at 16 Hz and 2.45 GHz radiation separately for a period of 14 days. There was no tumor development in mice exposed to RF and MW. Similarly a topical application of single dose of DMBA followed by RF/MW exposure also did not produce any visible extra tumor on the skin of mice. On the other hand mice were transplanted intraperitoneally with ascites (8 × 10(8) cell/ml) and subsequently exposed to above mentioned fields for 14 days showed a slight increase in the cell numbers as compared to the control group. However, the increase is insignificant. There were insignificant differences either in the mortality or cell proliferation among the control and exposed group. This results show that low level RF or MW do not alter tumor growth and development as evidenced by no observable change in tumor size.     

Inhibition of Lewis lung carcinoma in mice by local microwave hyperthermia combined with immunomodulating Propionibacterium granulosum KP-45

Summary C-57 BL/6 mice with Lewis lung carcinoma were treated 2 weeks after tumor implantation with local microwave hyperthermia (2450 MHz, tumor temperature 43.5° C, 30 min) and/or intratumoral or intraperitoneal injection of 1 mg cell walls of Propionibacterium granulosum KP-45. Tumor growth, survival time of the animals, and the delayed skin hypersensitivity to oxazolone were followed up, as well as the ³H-thymidine uptake of tumorous tissues and the number of lung metastases. The combined treatment of microwave hyperthermia with immunomodulating P. granulosum KP-45 resulted in significantly stronger inhibition of tumor growth than with each of these methods alone. The number of lung metastases could be significantly lowered, and the skin reactivity to oxazolone remained enhanced during the whole observation period (over 70 days after tumor implantation). The implications of the test observation are discussed.     

Laser hyperthermia of tumors with the use of golden nanoparticles under the control of optical coherent tomography and acoustothermometry

The local laser hyperthermia of an experimental tumor RShM-5 of mice with the use of golden plasmin resonance nanoparticles has been carried out. The accumulation of particles in the tumor was controlled by the in vivo noninvasive method of optical coherent tomography. Using this method, the time of the maximum content of nanoparticles in the tumor was determined to be 5 h after the intravenous administration during which the laser hyperthermia was performed. The control of the tumor temperature during the hyperthermia seance showed that the application of nanoparticles provides an effective temperature elevation inside the node and a more targeted heating. The local laser hyperthermia with nanoparticles induced the inhibition of the tumor growth from day 5 to day 7 after the seance with a maximum value of 56%.     

热疗对SW1990胰腺癌细胞上皮间质转化（EMT）的影响及其作用机制

目的：探讨热疗对化疗诱导的人胰腺癌细胞株SW1990细胞上皮间质转化（EMT）的影响及其可能的作用机制。方法：分别用不同浓度吉西他滨（0,5,10,20,30wM）作用于SW1990细胞不同时间（24,48,72小时）及30μM吉西他滨作用24h联合热疗43℃1h,观察细胞的形态学变化,通过四甲基偶氮唑蓝（MTT）法检测细胞的增殖情况。采用Westernblot方法检测细胞内E-cadherin蛋白和剪切的Notchl蛋白的表达。结果：①吉西他滨在一定浓度范围内可明显抑制SW1990细胞的增殖（P〈0．05）,并呈浓度和时间依赖性。吉西他滨作用前24h给予43℃1h热疗预处理可显著增强吉西他滨对SW1990细胞的抑制作用（P〈0．05）②吉西他滨作用SW1990细胞24小时后,细胞数目减少,细胞形态变大,细胞呈梭形,且细胞间连接减少;而热疗预处理的联合能够逆转此种形态学变化。③吉西他滨作用24小时后,细胞内E-cadherin蛋白的表达下调、CleavedNotchl的蛋白表达上调,热疗预处理可明显上调吉西他滨诱导的E-cadherin蛋白表达下调、并下调CleavedNotchl蛋白表达的上调。结论：热疗预处理显著逆转吉西他滨所诱导的人胰腺癌细胞株sW1990细胞的EMT现象,其机制可能与Notch信号通路有关。     

A miRNA Signature of Chemoresistant Mesenchymal Phenotype Identifies Novel Molecular Targets Associated with Advanced Pancreatic Cancer

In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.     

Relevance of biopsy-derived pancreatic organoids in the development of efficient transcriptomic signatures to predict adjuvant chemosensitivity in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) patients are frequently treated by chemotherapy. Even if personalized therapy based on molecular analysis can be performed for some tumors, PDAC regimens selection is still mainly based on patients' performance status and expected efficacy. Therefore, the establishment of molecular predictors of chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. We have recently developed an RNA-based signature that predicts the efficacy of adjuvant gemcitabine using 38 PDAC primary cell cultures. While demonstrated its efficiency, a significant association with the classical/basal-like PDAC spectrum was observed. We hypothesized that this flaw was due to the basal-like biased phenotype of cellular models used in our strategy. To overcome this limitation, we generated a prospective cohort of 27 consecutive biopsied derived pancreatic organoids (BDPO) and include them in the signature identification strategy. As BDPO's do not have the same biased phenotype as primary cell cultures we expect they can compensate one with each other and cover a broader range of molecular phenotypes. We then obtained an improved signature predicting gemcitabine sensibility that was validated in a cohort of 300 resected PDAC patients that have or have not received adjuvant gemcitabine. We demonstrated a significant association between the improved signature and the overall and disease-free survival in patients predicted as sensitive and treated with adjuvant gemcitabine. We propose then that including BDPO along primary cell cultures represent a powerful strategy that helps to overcome primary cell cultures limitations producing unbiased RNA-based signatures predictive of adjuvant treatments in PDAC.     

Ultrasound-mediated microbubbles cavitation enhanced chemotherapy of advanced prostate cancer by increasing the permeability of blood-prostate barrier

Although chemotherapy is an important treatment for advanced prostate cancer, its efficacy is relatively limited. Ultrasound-induced cavitation plays an important role in drug delivery and gene transfection. However, whether cavitation can improve the efficacy of chemotherapy for prostate cancer remains unclear. In this study, we treated RM-1 mouse prostate carcinoma cells with a combination of ultrasound-mediated microbubble cavitation and paclitaxel. Our results showed that combination therapy led to a more pronounced inhibition of cell viability and increased cell apoptosis. The enhanced efficacy of chemotherapy was attributed to the increased cell permeability induced by cavitation. Importantly, compared with chemotherapy alone (nab-paclitaxel), chemotherapy combined with ultrasound-mediated microbubble cavitation significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice in an orthotopic mouse model of RM-1 prostate carcinoma, indicating the synergistic effects of combined therapy on tumor reduction. Furthermore, we analyzed tumor-infiltrating lymphocytes and found that during chemotherapy, the proportions of CTLA4⁺ cells and PD-1⁺/CTLA4⁺ cells in CD8⁺ T cells slightly increased after cavitation treatment.     

miR-211 Modulates Gemcitabine Activity Through Downregulation of Ribonucleotide Reductase and Inhibits the Invasive Behavior of Pancreatic Cancer Cells

Only a subset of radically-resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from gemcitabine-based chemotherapy, thus the identification of novel prognostic factors is essential. In a high-throughput, microRNA (miRNA) array, miR-211 emerged as the best discriminating miRNA, with high expression associated with long survival. Here, we further explored the biological role of miRNA-211 in gemcitabine activity in the human PDAC cells (SUIT-2) subclones SUIT2-007 and SUIT2-028. Our results showed that miR-211 was expressed differentially in PDAC cells characterized by differential metastatic capability. In particular, S2-028 with lower metastatic ability had a higher expression of miR-211, compared to the S2-007 with higher metastatic capacity. Enforced expression of miR-211 via pre-miR-211 significantly reduced cell migration and invasion (e.g., 40% reduction of invasion of SUIT2 cells, compared to control; p <.05). Moreover, we demonstrated that induction of the miR-211 expression in the cells increased the sensitivity to gemcitabine and reduced the expression of its target ribonucleotide reductase subunit 2 (RRM2). In conclusion, miR-211 functional analyses suggested the role of RRM2 as a target of miR-211 in the modulation of gemcitabine sensitivity. Moreover, inhibition of cell migration and invasion might explain the less aggressive behavior of pancreatic cancer cells with higher expression levels of miR-211.