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Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer.Graphical abstract: SENTI-101 schematic and mechanism of actionSENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow–derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

Supplementary Figure1 shows MSC homing to tumor and other organs in vivo using a bioluminescent reporter. Supplementary Figure 2. Characterization of tumor immune infiltrate of syngeneic preclinical intraperitoneal tumor models CT26-ip and B16F10-ip using IHC and flow cytometry. Supplementary Figure 3. Kaplan Meier Survival in B16F10-ip model comparing multiple combinations of effector cytokines expressed by MSCs. Supplementary Figure 4. Shows data supporting the construct optimization for SENTI-101 including promoter, orientation and signal sequence changes. Supplementary Figure 5. IL-21 functional validation using UO2S IL-21 receptor dimerization reporter assay. Supplementary Table 1. Shows the DNA sequence of SB00880, the lentiviral construct used to engineer SENTI-101. Supplementary Figure 6. Shows the SENTI-101 manufacturing process overview. Supplementary Figure 7. Shows supporting data for the study of pharmacokinetics and pharmacodynamics of SENTI-101. Supplementary Figure 8. Shows individual tumor burden for each mouse measured by bioluminescence imaging (BLI) after treatment with SENTI-101 or controls. Supplementary Figure 9. Percent change in mouse body weight after treatment. Supplementary Figure 10. Shows the controls and supporting data for the selective depletion of immune cell types. Supplementary Figure 11. Characterization of tumor infiltrating lymphocytes (TILs) in the B16F10-ip tumor model after treatment with mSENTI-101. Supplementary Figure 12. Anti-tumor immune response induced by SENTI-101 in CT-26ip model. Supplementary Figure 13. Shows individual tumor burden for each mouse measured by BLI after treatment with SENTI-101 and in combination with checkpoint inhibitor anti-PD-1.

Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer. Graphical abstract: SENTI-101 schematic and mechanism of action SENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow–derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

CD33 and FLT3 are validated targets for myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). These malignancies have poor prognosis and high clinical unmet need, and current targeted therapies present ample limitations. One challenge is that untargeted leukemic stem cells (LSCs) can contribute to eventual relapse; however, targeting the LSC markers FLT3 and CD33 can contribute to off-tumor toxicities against normal hematopoietic stem cells and progenitor cells (HSCs/HPCs), leading to bone marrow toxicities such as thrombocytopenia, neutropenia, and infectious complications. SENTI-202 is designed to address these challenges and provide a broader therapeutic window. SENTI-202 is a preclinical CAR-NK cell product candidate engineered with an OR and NOT logic gated gene circuit onto allogeneic healthy adult peripheral blood NK cells along with multi-arming via expression of calibrated release IL-15 (crIL-15) to enhance therapeutic activity and tolerability. The OR gate is implemented using a bivalent CAR (aCAR) that binds both CD33 and FLT3 to target both AML blasts and LSCs. Additionally, the NOT gate is implemented using an inhibitory CAR (iCAR) that recognizes the healthy cell protective antigen endomucin (EMCN) and targets endomucin which is designed to prevent CAR-mediated cytotoxicity against healthy cells, such as HSCs and early HPCs, potentially reducing on-target/off-tumor toxicities. Finally, crIL-15 provides NK cell activation and persistence via simultaneous autocrine, juxtracrine, and paracrine activities. Preclinical studies demonstrated the [antitumor] activity and tolerability of SENTI-202. All SENTI-202 components are stably expressed via a single g-retroviral vector. SENTI-202 preclinical pharmacokinetics and secondary pharmacodynamics demonstrated CAR target engagement, function, as well as mechanism of action for all components in different AML models. SENTI-202 CAR-NK cells demonstrated cytotoxic activity when co-cultured with AML target cells and primary patient-derived samples and had increased antitumor capacity compared to non-engineered NK cells. SENTI-202 induced the production of cytotoxic cytokines and increased expression of activation and cytotoxicity markers consistent with CAR-mediated activation of the NK cells. The presence of EMCN iCAR reduced cytotoxicity in an EMCN-dependent manner in target cells and primary HSCs. crIL-15 expression yielded functional pSTAT5 signaling and increased persistence of SENTI-202 in vitro and in vivo. These results demonstrate the antitumor activity and tolerability of SENTI-202 and warrant further clinical development as a novel therapeutic option for patients with CD33+ and/or FLT3+ tumors. Citation Format: Alba Gonzalez, Enping Hong, Gozde Yucel, Elizabeth Leitner, Pearley Chinta, Han Deng, Ian Li, Alice Lam, Abla Bakir, Brandon Lee, Papia Chakraborty, Carmina Blanco, Chen-Ting Lee, Niran Almudhfar, Mengxi Tian, Wenqi Song, Andrew Banicki, Otto Contreras, Martin Gieldin, Brian Garrison, Timothy K. Lu, Kanya Rajangam. Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3195.

Background: Chaperon-mediated autophagy (CMA) is a target specific degradation pathway among autophagic processes. Although CMA plays critical roles in tumor progression in general, the role of CMA in tumor progression under hypoxia is poorly understood. We investigated the role of CMA in hypoxic tumor using a novel Hsp90-mediated modulator of CMA.Methods: We examined whether manassantin A (ManA), known as a potent inhibitor of HIF-1α, is a CMA modulator using biochemical, molecular, and cell biology approaches. We analyzed the effects of ManA on Hsp90 chaperone function by using Significant Analysis of Microarray, luciferase refolding assay, HS-10 resin binding assay, NMR spectroscopy, and SPR assay. We investigated tumor growth in response to monotherapy and combination therapy with ManA and anti-programmed death-1 (PD-1) antibody in vivo. To assess the clinical efficacy of CMA-related genes, we analyzed the gene expressions of HIF-1α, HSP90AA1, and transcription factor EB (TFEB) using TCGA datasets. Finally, we assessed in vivo/in vitro absorption, distribution, metabolism, and excretion properties of ManA.Results: ManA inhibits Hsp90 chaperone function through disruption of the Hsp90/F1F0-ATP synthase (chaperone/co-chaperone) complex. The inhibition of Hsp90 enhances the interaction of CMA substrates and LAMP-2A as well as TFEB nuclear localization, thus leading to CMA activation. Importantly, CMA activation not only retards tumor growth in vitro and in vivo, but also displays cooperative antitumor activity with anti-PD-1 antibody in vivo. An in-depth analysis of TCGA datasets shows that combined expression of HSP90AA1High/HIF1AHigh or TFEBLow/HIF1AHigh is strongly correlated with poor prognosis in lung cancer patients. Conclusions: ManA-induced inhibition of Hsp90 promotes CMA activity and decreases the stability of CMA substrates such as HIF-1α protein, leading to a marked reduction of hypoxic tumor growth. Therefore, targeting CMA activity via Hsp90 may present a promising therapeutic strategy for hypoxic tumor.

BackgroundCAR-based therapies have transformed the treatment of several cancers, but this progress has not translated into solid tumors. One challenge of CAR-mediated therapies for solid tumors is the lack of specific tumor-associated antigens (TAA’s) that are only expressed on cancer cells and not on healthy cells, thereby posing a risk for on-target off-tumor toxicities. This presents a unique opportunity to use Logic Gates to expand the universe of cancer targets that may be treated with CAR-based cell therapies. CEA, a widely expressed tumor antigen, found in >90% of colorectal cancer (CRC), is also expressed in healthy gastrointestinal and lung epithelial cells. Clinical experience targeting CEA resulted in severe dose-limiting toxicities,1,2 highlighting the need for healthy tissue protection. Logic-gated gene circuits can prevent off-tumor toxicities by pairing a CEA activating-CAR (aCAR) with an inhibitory-CAR (iCAR) that recognizes a safety antigen (SA) uniquely expressed in healthy epithelial cells.MethodsWe developed a bioinformatics-driven antigen paired discovery platform using single-cell transcriptomics to discover and prioritize TAA’s and pair them with SA’s that are selectively expressed on the membrane of healthy cells. TAA’s and SA’s were validated in primary cancer and healthy tissue samples using IHC. We constructed aCAR/iCAR gene circuits and tested their function in NK cells.ResultsOur bioinformatics platform identified VSIG2 to be co-expressed with CEA in healthy gastrointestinal and lung epithelial cells. IHC confirmed the expression of VSIG2 on the membrane of healthy colon (N=72 samples) and lung (N=24 samples) epithelial cells.Using our Design-Build-Test-Learn platform, we screened >250 CAR constructs targeting CEA. CAR-NK cells were generated and tested for anti-tumor activity against CRC CEA+ cells and lead candidates were selected based on NK cell performance. A single dose of CEA-CAR-NK cells had anti-tumor activity in a human CRC xenograft model, reducing tumor burden in >33% of the treated mice. We identified iCARs with different intracellular domains derived from native domains containing immunoreceptor tyrosine-based inhibitory motifs. These iCARs suppressed >50% of aCAR-mediated killing (pConclusionsWe are developing Logic-Gated CAR-NK cell therapies aimed at reducing on-target off-tumor toxicities, to spare healthy cells in a SA-dependent manner. SENTI-401 will focus on targeting CEA+ CRC tumors with a NOT gate that recognizes the SA VSIG2 in the colon and lungs.ReferencesParkhurst M, et al. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011; Mar;19(3):620–6.Thistlethwaite FC, et al. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother 2017 Nov;66(11):1425–1436.

Metastatic spread is the mechanism in more than 90 percent of cancer deaths and current therapeutic options, such as systemic chemotherapy, are often ineffective. Here we provide a proof of principle for a novel two-pronged modality referred to as Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) having the potential to safely eradicate both primary tumors and distant metastatic foci. Using a combination of immune-checkpoint inhibition and plasmonic gold nanostar (GNS)–mediated photothermal therapy, we were able to achieve complete eradication of primary treated tumors and distant untreated tumors in some mice implanted with the MB49 bladder cancer cells. Delayed rechallenge with MB49 cancer cells injection in mice that appeared cured by SYMPHONY did not lead to new tumor formation after 60 days observation, indicating that SYMPHONY treatment induced effective long-lasting immunity against MB49 cancer cells.

In 2011 Hanahan and Weinberg updated their well-established paper 'The hallmarks of cancer'. The rationale for that review and its predecessor was to produce a conceptual framework for future research in cancer. The original Hallmarks included: cell signalling to enhance tumour cell proliferation, acquisition of ability to evade growth suppressors, developing mechanisms to resist cell death, enabling replicative immortality, initiating angiogenesis and activating processes to enable invasion and metastasis. In the more recent paper, Hanahan and Weinberg added important new features to this composite paradigm. The new features were: (1) altered metabolism, (2) evasion of immune destruction, (3) tumour promoting inflammation, and (4) the cellular microenvironment. These four new features are the main focus of this review. Hanahan and Weinberg did not specifically include the physiological microenvironment which is dominated by hypoxia and acidosis. In this review we will consider these features in addition to the cellular and metabolic components of the microenvironment. The purpose of this review is to present a vision of emerging fields of study in hyperthermia biology over the next decade and beyond. As such, we are focusing our attention on pre-clinical studies, primarily using mice. The application of hyperthermia in human patients has been thoroughly reviewed elsewhere.

While immunotherapies based on single recombinant cytokines such as IL12 and IL21 have shown great promise in preclinical models of solid tumors, clinical translation has proven challenging due to limited mechanisms of action, narrow therapeutic windows upon systemic administration, and short half-lives resulting in poor pharmacokinetics and distribution. Thus, there is a need for tumor-localized cytokine therapies capable of driving sustained efficacy with a wide therapeutic window. SENTI-101 is a cell-based immunotherapy comprising allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) genetically modified to express IL12 and IL21. Consistent with prior studies, we demonstrated that SENTI-101 innately homes to disseminated tumors in the peritoneal cavity and induces durable anti-tumor responses and immune memory in various preclinical models of peritoneal tumors. In this study, we investigated the mechanisms of action of SENTI-101. Our results demonstrate that the IL12 and IL21 combination elicits pleiotropic and complementary effects that drive a multi-modal immune response across various steps of the cancer immunity cycle. Treatment of preclinical murine models of peritoneal tumors (e.g., CT26 and B16F10) with SENTI-101 significantly increased the local production of IFNg by more than 40-fold (p Overall, our preclinical studies show that SENTI-101 modulates the tumor immune landscape via multiple complementary modes of action, resulting in long-term anti-tumor immunity. Furthermore, this work demonstrates the therapeutic potential of tumor-localized cell therapies armed with gene circuits expressing combinatorial immune effectors to trigger a multi-factorial anti-tumor response. Citation Format: Alba Gonzalez, Frances D. Liu, Archana Nagaraja, Alyssa Mullenix, Russell M. Gordley, Daniel O. Frimannsson, Anissa Benabbas, Chen-Ting Lee, Tiffany A. Truong, Allison Quach, Mengxi Tian, Rowena Martinez, Rishi Savur, Alyssa Perry-McNamara, Don-Hong Wang, Ori Maller, Dharini Iyer, Ashita Magal, Sravani Mangalampalli, Christina J. Huynh, Carmina C. Blanco, Jack T. Lin, Brian S. Garrison, Philip Lee, Timothy K. Lu, Gary Lee. SENTI-101, a novel genetically modified allogeneic cell product expressing IL12 and IL21, elicits a tumor-localized, robust, and multimodal immune response in preclinical models of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4246.

To ensure reliability and reproducibility of radiobiological data, it is necessary to standardize dosimetry practices across all research institutions. The photoelectric effect predominates over other interactions at low energy and in high atomic number materials such as bone, which can lead to increased dose deposition in soft tissue adjacent to mineral bone due to secondary radiation particles. This may produce radiation effects that deviate from higher energy photon irradiation that best model exposure from clinical radiotherapy or nuclear incidences. Past theoretical considerations have indicated that this process should affect radiation exposure of neighboring bone marrow (BM) and account for reported differences in relative biological effectiveness (RBE) for hematopoietic failure in rodents. The studies described herein definitively estimate spatial dose distribution and biological effectiveness within the BM compartment for (137)Cs gamma rays and 320 kVp X rays at two levels of filtration: 1 and 4 mm Cu half-value layer (HVL). In these studies, we performed: 1. Monte Carlo simulations on a 5 μm resolution model of mouse vertebrae and femur derived from micro-CT images; 2. In vitro biological experiments irradiating BM cells plated directly on the surface of a bone-equivalent material (BEM); and 3. An in vivo study on BM cell survival in irradiated live mice. Simulation results showed that the relative dose increased in proximity to bone at the lower radiation energies and produced averaged values of relative dose over the entire BM volume within imaged trabecular bone of 1.17, 1.08 and 1.01 for beam qualities of 1 mm Cu HVL, 4 mm Cu HVL and (137)Cs, respectively. In accordance with Monte Carlo simulations, in vitro irradiation of BM cells located on BEM and in vivo whole-body irradiation at a prescribed dose to soft tissue of 6 Gy produced relative cell killing of hematopoietic progenitors (CFU-C) that significantly increased for the 1 mm Cu HVL X rays compared to radiation exposures of higher photon energies. Thus, we propose that X rays of the highest possible kVp and filtration be used to investigate radiation effects on the hematopoietic system, as this will allow for better comparisons with high-energy photon exposures applied in radiotherapy or as anticipated in a nuclear event.

To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin’s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers.

Normal tissue damage after head and neck radiotherapy involves a continuum of pathologic events to the mucosa, tongue and salivary glands. We examined the radioprotective effects of MnBuOE, a redox-active manganese porphyrin, at three stages of normal tissue damage: immediate (leukocyte endothelial cell [L/E] interactions), early (mucositis) and late (xerostomia and fibrosis) after treatment. In this study, mice received 0 or 9 Gy irradiation to the oral cavity and salivary glands ± MnBuOE treatment. Changes in leukocyte-endothelial cell interactions were measured 24 h postirradiation. At 11 days postirradiation, mucositis was assessed with a cathepsin-sensitive near-infrared optical probe. Stimulated saliva production was quantified at 11 weeks postirradiation. Finally, histological analyses were conducted to assess the extent of long-term effects in salivary glands at 12 weeks postirradiation. MnBuOE reduced oral mucositis, xerostomia and salivary gland fibrosis after irradiation. Additionally, although we have previously shown that MnBuOE does not interfere with tumor control at high doses when administered with radiation alone, most head and neck cancer patients will be treated with the combinations of radiotherapy and cisplatin. Therefore, we also evaluated whether MnBuOE would protect tumors against radiation and cisplatin using tumor growth delay as an endpoint. Using a range of radiation doses, we saw no evidence that MnBuOE protected tumors from radiation and cisplatin. We conclude that MnBuOE radioprotects normal tissue at both early and late time points, without compromising anti-tumor effects of radiation and cisplatin.

We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20-26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30-31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8(+) T lymphocytes and CD8(+) T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

This paper describes a preclinical investigation of the feasibility of thermotherapy treatment of bladder cancer with magnetic fluid hyperthermia (MFH), performed by analysing the thermal dosimetry of nanoparticle heating in a rat bladder model.The bladders of 25 female rats were instilled with magnetite-based nanoparticles, and hyperthermia was induced using a novel small animal magnetic field applicator (Actium Biosystems, Boulder, CO). We aimed to increase the bladder lumen temperature to 42 °C in10 min and maintain that temperature for 60 min. Temperatures were measured within the bladder lumen and throughout the rat with seven fibre-optic probes (OpSens Technologies, Quebec, Canada). An MRI analysis was used to confirm the effectiveness of the catheterisation method to deliver and maintain various nanoparticle volumes within the bladder. Thermal dosimetry measurements recorded the temperature rise of rat tissues for a variety of nanoparticle exposure conditions.Thermal dosimetry data demonstrated our ability to raise and control the temperature of rat bladder lumen ≥1 °C/min to a steady state of 42 °C with minimal heating of surrounding normal tissues. MRI scans confirmed the homogenous nanoparticle distribution throughout the bladder.These data demonstrate that our MFH system with magnetite-based nanoparticles provides well-localised heating of rat bladder lumen with effective control of temperature in the bladder and minimal heating of surrounding tissues.

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the Stability of Proteins from Rates of Oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a Stable Isotope Labeling with Amino acids in Cell culture strategy (SILAC-PP), over 1,000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A’s mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anti-cancer drug with an unknown mode-of-action.

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as “alternative therapies”. In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.

Breast cancer is the most common malignancy diagnosed among women and represents a heterogeneous group of subtypes. Radiation therapy is a critical component of treatment for breast cancer patients. However, little is known about radiation response among these intrinsic subtypes. In previous studies, we identified a significant induction of FAS after irradiation in biologically favorable breast cancer patients and breast cancer cell lines. Here, we expanded our study and investigated radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation to assess tumor growth control. We further investigated the therapeutic effect of FAS modulation by silencing FAS in radiation-responsive tumors and injecting FAS agonist antibody into radiation-resistant tumors. Exposure to radiation inhibited MCF7, and to a lesser extent HCC1954 tumor growth in a dose-dependent manner. In contrast, SUM159 tumors were resistant to radiation. The estimated TCD50 values were 19.3 Gy for MCF7 and 44.9 Gy for SUM159. Radiation induced FAS expression in MCF7 tumors, but not SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation treatment delayed SUM159 and HCC1954 tumor growth. However, it did not reach statistical significance compared to radiation treatment alone. Our results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS activation concurrent with radiation slows tumor growth in the radiation-resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation.

The increase in penicillin resistance among pneumococci and viridans streptococci and the development of serotype-specific conjugate vaccine have increased the need for knowledge of the antimicrobial susceptibility and the capsular serotypes of group B streptococci. Over an 8-year period, 351 group B streptococcal isolates from southern Taiwan were tested for antimicrobial susceptibility and serotype determination. Eighty-seven percent of the isolates were typeable. Types III (28.5%) and V (27.1%) were the most common serotypes. The occurrence of type V isolates increased with age, while that of type III isolates decreased with age, showing a predominance in children less than 1 year of age. Of 118 isolates from cases of invasive infection, types Ia, Ib, II, III, IV, and V accounted for 12.7, 11.9, 0.8, 33, 1.7, and 26.3%, respectively. Using the agar dilution method, all isolates were found to be susceptible to penicillin, cefotaxime, and vancomycin, 99.4% to ofloxacin, 78.1% to chloramphenicol, 63.2% to azithromycin, 62.6% to erythromycin, 57.3% to clindamycin, and 2.8% to tetracycline. Chloramphenicol resistance was associated with type III isolates (59 of 100, 59%) and erythromycin and azithromycin resistance with type Ib isolates (25 of 33 176%], and 21 of 33 [64%], respectively). Thus, 72% of the isolates from invasive infections were serotype III, V, or Ia, and penicillin remains the drug of choice for treatment or prophylaxis of group B streptococcal infections in southern Taiwan, despite the high prevalence of penicillin resistance among Streptococcus pneumoniae and viridans streptococci.

Significance: Most solid tumors contain regions of low oxygenation or hypoxia. Tumor hypoxia has been associated with a poor clinical outcome and plays a critical role in tumor radioresistance. Recent Advances: Two main types of hypoxia exist in the tumor microenvironment: chronic and cycling hypoxia. Chronic hypoxia results from the limited diffusion distance of oxygen, and cycling hypoxia primarily results from the variation in microvessel red blood cell flux and temporary disturbances in perfusion. Chronic hypoxia may cause either tumor progression or regressive effects depending on the tumor model. However, there is a general trend toward the development of a more aggressive phenotype after cycling hypoxia. With advanced hypoxia imaging techniques, spatiotemporal characteristics of tumor hypoxia and the changes to the tumor microenvironment can be analyzed. Critical Issues: In this review, we focus on the biological and clinical consequences of chronic and cycling hypoxia on radiation treatment. We also discuss the advanced non-invasive imaging techniques that have been developed to detect and monitor tumor hypoxia in preclinical and clinical studies. Future Directions: A better understanding of the mechanisms of tumor hypoxia with non-invasive imaging will provide a basis for improved radiation therapeutic practices. Antioxid. Redox Signal. 21, 313–337.

Despite positive efficacy, thermotherapy is not widely used in clinical oncology. Difficulties associated with field penetration and controlling power deposition patterns in heterogeneous tissue have limited its use for heating deep in the body. Heat generation using iron-oxide super-paramagnetic nanoparticles excited with magnetic fields has been demonstrated to overcome some of these limitations. The objective of this preclinical study is to investigate the feasibility of treating bladder cancer with magnetic fluid hyperthermia (MFH) by analyzing the thermal dosimetry of nanoparticle heating in a rat bladder model.The bladders of 25 female rats were injected with 0.4 ml of Actium Biosystems magnetite-based nanoparticles (Actium Biosystems, Boulder CO) via catheters inserted in the urethra. To assess the distribution of nanoparticles in the rat after injection we used the 7 T small animal MRI system (Bruker ClinScan, Bruker BioSpin MRI GmbH, Ettlingen, Germany). Heat treatments were performed with a small animal magnetic field applicator (Actium Biosystems, Boulder CO) with a goal of raising bladder temperature to 42°C in10min and maintaining for 60min. Temperatures were measured throughout the rat with seven fiberoptic temperature probes (OpSens Technologies, Quebec Canada) to characterize our ability to localize heat within the bladder target.The MRI study confirms the effectiveness of the catheterization procedure to homogenously distribute nanoparticles throughout the bladder. Thermal dosimetry data demonstrate our ability to controllably raise temperature of rat bladder ≥1°C/min to a steady-state of 42°C.Our data demonstrate that a MFH system provides well-localized heating of rat bladder with effective control of temperature in the bladder and minimal heating of surrounding tissues.

Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio. Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p = NS). In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation. Citation Format: Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1656.

Background: Breast cancer is the most common female malignancy in the US, and radiotherapy is a routine part of multi-disciplinary breast cancer care. Radiation treatment (RT) tailored to target radiation response pathways could maximize treatment benefit in radiation resistant tumors, while minimizing unnecessary toxicity and cost in more radiation responsive breast tumor subtypes. Here we report on genome-wide analysis, gene expression profiles and biological pathways associated with RT response in breast cancer patients. Methods: Between 2010-2013, a total of 32 breast cancer patients were enrolled at Duke University Medical Center on an IRB approved Phase I clinical trial testing preoperative radiotherapy. Paired pre and post-RT FFPE biopsy samples were available for N = 26. Genome wide-analysis was performed using two distinct techniques: i) Microarray analysis using the Affymetrix GeneChip ® Human Transcriptome Array 2.0 (HTA 2.0); ii) RNA-Seq using TruSeq ®RNA Access Library kit (Illumina). All libraries were sequenced on the Illumina HiSeq 2000 generating 50 bp single reads at a sequencing depth of 40 million reads. The RNA-Seq data was processed using the TrimGalore toolkit. For the paired pre and post-RT differential gene expression analysis we used the linear model. Genes that had an FDR ≤ 5% in the RNA-Seq experiment were compared with all microarray probesets that had an FDR ≤ 5%. If at least one probeset that belonged to a gene in the RNA-Seq list was significant and showed the same direction of change between pre and post-RT samples, they were identified as consistent. Gene Set Enrichment Analysis (GSEA) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to identify significant pathways and gene ontologies that were differentially regulated between the conditions. Results: 2648 genes showed the same significant results in response to RT in microarray and RNA-Seq including: i) a total of 1401 genes that had significantly higher expression levels, and ii) a total of 1247 genes that had significantly lower expression levels following RT. Principal Components Analysis (PCA) demonstrated distinct separation of pre and post-RT gene expression signatures. Differentially expressed genes were involved in major biological pathways such as cell cycle checkpoint, DNA replication and DNA repair pathways. A subset of these genes also demonstrated differential expression in radiation responsive vs. non-responsive breast cancer cell lines. Conclusion: New laboratory techniques have become available which provide the opportunity to use FFPE samples for microarray and RNA-seq analysis. Using these new techniques and our rare pre and post-RT FFPE breast tumor samples, we have been able to demonstrate distinct differences in gene expression profiles and modulation of biological pathways in response to RT in human breast tumors. Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, David Corcoran, Chen-Ting Lee, Ying Zhou, Mark W. Dewhirst, Jen-Tsan A. Chi, Janet K. Horton. Radiation response genome-wide analysis using paired pre and post-radiation FFPE human breast tumor samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1647.

Macrophages function both under normothermia and during periods of body temperature elevation (fever). Whether macrophages sense and respond to thermal signals in a manner which regulates their function in a specific manner is still not clear. In this brief review, we highlight recent studies which have analyzed the effects of mild heating on macrophage cytokine production, and summarize thermally sensitive molecular mechanisms, such as heat shock protein (HSP) expression, which have been identified. Mild, physiologically achievable, hyperthermia has been shown to have both pro- and anti-inflammatory effects on macrophage inflammatory cytokine production and overall it is not clear how hyperthermia or HSPs can exert opposing roles on macrophage function. We propose here that the stage of activation of macrophages predicts how they respond to mild heating and the specific manner in which HSPs function. Continuing research in this area is needed which will help us to better understand the immunological role of body temperature shifts. Such studies could provide a scientific basis for the use of heat in treatment of inflammatory diseases.

Macrophages are often considered the sentries in innate immunity, sounding early immunological alarms, a function which speeds the response to infection. Compared to the large volume of studies on regulation of macrophage function by pathogens or cytokines, relatively little attention has been devoted to the role of physical parameters such as temperature. Given that temperature is elevated during fever, a long-recognized cardinal feature of inflammation, it is possible that macrophage function is responsive to thermal signals. To explore this idea, we used LPS to model an aseptic endotoxin-induced inflammatory response in BALB/c mice and found that raising mouse body temperature by mild external heat treatment significantly enhances subsequent LPS-induced release of TNF-α into the peritoneal fluid. It also reprograms macrophages, resulting in sustained subsequent responsiveness to LPS, i.e., this treatment reduces "endotoxin tolerance" in vitro and in vivo. At the molecular level, elevating body temperature of mice results in a increase in LPS-induced downstream signaling including enhanced phosphorylation of IKK and IκB, NF-κB nuclear translocation and binding to the TNF-α promoter in macrophages upon secondary stimulation. Mild heat treatment also induces expression of HSP70 and use of HSP70 inhibitors (KNK437 or Pifithrin-µ) largely abrogates the ability of the thermal treatment to enhance TNF-α, suggesting that the induction of HSP70 is important for mediation of thermal effects on macrophage function. Collectively, these results support the idea that there has been integration between the evolution of body temperature regulation and macrophage function that could help to explain the known survival benefits of fever in organisms following infection.

In this study, we asked whether exposure to different physiologically relevant temperatures (33°C, 37°C, and 39.5°C) could affect subsequent antigen-specific, activation-related events of naive CD8+ T cells. We observed that temporary exposure of CD62LhiCD44lo Pmel-1 CD8+ cells to 39.5°C prior to their antigen-dependent activation with gp10025–33 peptide-pulsed C57BL/6 splenocytes resulted in a greater percentage of cells, which eventually differentiated into CD62LloCD44hi effector cells compared with cells incubated at 33°C and 37°C. However, the proliferation rate of naive CD8+ T cells was not affected by mild heating. While exploring these effects further, we observed that mild heating of CD8+ T cells resulted in the reversible clustering of GM1+ CD-microdomains in the plasma membrane. This could be attributable to a decrease in line tension in the plasma membrane, as we also observed an increase in membrane fluidity at higher temperatures. Importantly, this same clustering phenomenon was observed in CD8+ T cells isolated from spleen, LNs, and peripheral blood following mild whole-body heating of mice. Further, we observed that mild heating also resulted in the clustering of TCRβ and the CD8 coreceptor but not CD71R. Finally, we observed an enhanced rate of antigen-specific conjugate formation with APCs following mild heating, which could account for the difference in the extent of differentiation. Overall, these novel findings may help us to further understand the impact of physiologically relevant temperature shifts on the regulation of antigen-specific CD8+ T cell activation and the subsequent generation of effector cells.

Macrophages create a major danger signal following injury or infection and upon activation release pro-inflammatory cytokines, which in turn help to generate febrile conditions. Thus, like other cells of the body, tissue macrophages are often exposed to naturally occurring elevations in tissue temperature during inflammation and fever. However, whether macrophages sense and respond to temperature changes in a specific manner which modulates their function is still not clear. In this brief review, we highlight recent studies which have analyzed the effects of temperatures on macrophage function, and summarize the possible underlying molecular mechanisms which have been identified. Mild, physiological range hyperthermia has been shown to have both pro- and anti-inflammatory roles in regulating macrophage inflammatory cytokine production and at the meeting presentation, we will show new data demonstrating that hyperthermia can indeed exert both positive and negative signals to macrophages. While some thermal effects are correlated with the induction of heat shock factors/heat shock proteins, overall it is not clear how mild hyperthermia can exert both pro- and anti-inflammatory functions. We also summarize data which shows that hyperthermia can affect other macrophage effector functions, including the anti-tumor cytotoxicity. Overall, these studies may help us to better understand the immunological role of tissue temperature and may provide important information needed to maximize the application of heat in the treatment of various diseases including cancer.

Hypoxia within the tumour microenvironment is correlated with poor treatment outcome after radiation and chemotherapy, and with decreased overall survival in cancer patients. Several molecular mechanisms by which hypoxia supports tumour growth and interferes with effective radiation and chemotherapies are now well established. However, several new lines of investigation are pointing to yet another ominous outcome of hypoxia in the tumour microenvironment: suppression of anti-tumour immune effector cells and enhancement of tumour escape from immune surveillance. This review summarises this important information, and highlights mechanistic data by which hypoxia incapacitates several different types of immune effector cells, enhances the activity of immunosuppressive cells and provides new avenues which help 'blind' immune cells to detect the presence of tumour cells. Finally, we discuss data which indicates that mild thermal therapy, through its physiologically regulated ability to alter vascular perfusion and oxygen tensions within the tumour microenvironment, as well as its ability to enhance the function of some of the same immune effector activities that are inhibited by hypoxia, could be used to rapidly and safely release the tight grip of hypoxia in the tumour microenvironment thereby reducing barriers to more effective immune-based therapies.

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in radioresistant basal cell lines. Our findings suggest that cell-type-specific, radiation-induced FAS contributes to subtype-specific breast cancer radiation response and that activation of FAS pathways may be exploited for biologically tailored radiotherapy.

Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, breast radiotherapy is currently delivered uniformly, regardless of breast cancer subtype. Recent clinical data have suggested that different subtypes of breast cancer may respond differently to radiation. Understanding the intrinsic variation of radiation response in breast cancer subtypes would lay the foundation for individualized radiotherapy designed to limit toxicity in those with radiation responsive cancers. Furthermore, identification of molecular targets can be used to modulate treatment outcomes in patients with radiation resistant tumors. Our previous in vitro studies have identified an association between breast cancer subtype and gene expression profiles in response to radiation in breast tumor cell lines. Cell lines with the greatest response to radiation were luminal subtype and exhibited preferential induction of genes involved in programmed cell death, such as FAS. In this study, we investigated tumor growth and FAS induction in response to radiation in a mouse model of breast cancer. MCF7 (luminal) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of athymic nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7 tumor growth in a dose-dependent manner, while radiation treatment had little effect on SUM159 tumor growth. In addition, FAS was induced in MCF7 tumors 24 hour after radiation, but there was no change in FAS expression in SUM159 tumors. However, injection of FAS-stimulating antibody intratumorally led to downstream caspase 8 and PARP activation, suggesting that the FAS signaling pathway remains intact and can be used to modulate radiation response in the resistant SUM159 cells. In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes and that stimulation of FAS may have the potential to modulate radiation response. Citation Format: Chen-Ting Lee, Yingchun Zhou, Sharareh Siamakpour-Reihani, Kingshuk R. Choudhury, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in a mouse model of human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2015-3302

Background: Breast radiotherapy is currently “one size fits all” regardless of breast cancer subtype (eg. luminal, basal). Emerging clinical data suggests that these distinct subtypes of breast cancer have unique patterns of response to radiation. There is a clinical need to identify radiation response biomarkers in order to provide individualized and optimal radiotherapy. Here we report on gene expression profiles associated with radiation response from: i) a rare clinico-genomic data in women treated with preoperative breast radiation and, ii) data from a pilot study of 16 biologically diverse breast tumor cell lines. Methods: Microarray analysis was done using the Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) and the Affymetrix HU133A2.0 arrays. RNA was harvested from paired pre- and post-radiation formalin-fixed paraffin-embedded breast cancer tissue in 26 patients treated with radiation prior to surgical resection. In addition, RNA was obtained from 16 biologically diverse breast cancer cell lines exposed to radiation treatment (5Gy and 0Gy). The genes with the highest coefficient of variation were selected and a paired sample t-test was used to evaluate the significance of these changes. We compared the top 100 significantly induced human genes with genes identified as differentially expressed in the 16 breast cancer cell lines. Findings of interest were validated using qPCR, IHC and wWestern blotting assays. Results: Fourteen genes had significant adjusted p-values for differential expression in the human data and were differentially expressed among the radiation responsive versus non-responsive cell lines. Differential expression of genes involved in: apoptosis, cell cycle and MAPK signaling pathways were observed in both the luminal human tumors and the largely luminal radiation responsive cell lines. Differential expression of FAS, a critical modulator of programmed cell death, was highly significant. Conclusion: The known function of FAS suggests a biologically plausible role in breast subtype specific radiation response. Induction of FAS is seen in human tissue, and preferentially in luminal breast cancer cell lines. Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, Chen-Ting Lee, Yingchun Zhou, Kouros Owzar, Jen-Tsan Chi, Janet K. Horton. Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3331. doi:10.1158/1538-7445.AM2015-3331

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Current standard treatment for GBM following maximum safe resection includes temozolomide (TMZ) with concurrent radiotherapy. Despite the improvement of survival by TMZ and radiation, the overall prognosis remains poor for GBM patients. This is partially attributed to drug resistance. Expression of DNA repair protein O6-methylguanine methyltransferase (MGMT) in gliomas confers TMZ resistance and a worse prognosis. Therefore, development of novel therapy to suppress MGMT expression and overcome TMZ resistance is highly desired. Hyperthermia has been shown to increase TMZ sensitivity in melanoma and this provides a good rationale to evaluate whether hyperthermia overcomes TMZ resistance in glioblastoma cells. In this study, we investigated the effect of hyperthermia treatment (43 °C) on TMZ sensitivity of four human glioblastoma cell lines using clonogenic assays. Our results showed that hyperthermia treatment increased TMZ-induced cell death in TMZ-resistant glioma cells, but had no additional beneficial effect in the TMZ-sensitive cells. Hyperthermia in combination with radiation further overcame TMZ resistance. To study the molecular targets, we found that hyperthermia downregulated MGMT expression through increasing proteasome-mediated MGMT degradation, which can be reversed by the proteasome inhibitor bortezomib. Hyperthermia had no effects on p53 expression and MGMT promoter methylation which was responsible for MGMT gene silencing. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed an increase of TMZ and its downstream product 5-aminoimidazole-4-carboxamide (AIC) in hyperthermia-treated cells, indicating an increase of TMZ drug uptake and breakdown. In conclusion, these results suggest that hyperthermia overcomes MGMT-mediated resistance of glioblastoma cell lines to TMZ and increases TMZ drug uptake. This study suggests a potential benefit of using hyperthermia in the treatment of GBM, particularly those with more adverse prognosis. This work was supported by Debrule Research Fund. Citation Format: Chen-Ting Lee, Aaron Blackley, Chelsea Landon, Ivan Spasojevic, John P. Kirkpatrick, Mark W. Dewhirst. Hyperthermia treatment overcomes temozolomide resistance in glioma cells by downregulating MGMT expression and increasing temozolomide uptake. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2014-3774

A total of 204 nonrepetitive isolates of group A streptococci (GAS), including 107 randomly collected between 1992 and 1995 and 66 and 31 consecutively collected in 1997 and 1998, respectively, from a university hospital in southern Taiwan were examined to determine the prevalence and mechanisms of erythromycin resistance among these isolates. Resistance to erythromycin was detected in 129 isolates (63.2%) by the agar dilution test. Of these, 42 isolates (32.6%) were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and all carried the ermB gene; 4 (3.1%) were assigned to the inducible MLS resistance (iMLS) phenotype, and all harbored the ermTR gene; and 83 (64.3%) were erythromycin resistant but susceptible to clindamycin (M phenotype), and all possessed the mefA gene. Distributed by years, the rates of erythromycin resistance and different phenotypes were 61.7% (53.0% cMLS, 6.1% iMLS, and 40.9% M phenotype) between 1992 and 1995, 62.1% (12.2% cMLS and 87.8% M phenotype) in 1997, and 71.0% (9.1% cMLS and 90.9% M phenotype) in 1998. Pulsed-field gel electrophoresis showed that all but 2 cMLS isolates were clonal in origin, and 17 clones were detected among the M-phenotype isolates. These results indicate that the high incidence and increasing rate of erythromycin-resistant GAS in southern Taiwan are due to the prevalence of multiple M-phenotype clones and that clindamycin may be the drug of choice for the treatment of infections with GAS in penicillin-hypersensitive patients in this area.

Laboratory mice are housed under relatively cool conditions and even though their core temperature remains normal, mice are cold stressed and exhibit significant increases in metabolic heat production. We compared tumor growth in mice housed at a standard ambient temperature (22oC) and at a thermoneutral temperature (30oC) which reduces metabolic effort needed for maintaining core temperature. We found significantly reduced tumor incidence and growth rates in animals housed at thermoneutral vs standard temperature using four different syngeneic tumor models, including a metastasis model. Because this difference was not seen in SCID or NUDE mice or in mice depleted of CD8+ T cells, we suspected that a prominent result of metabolic cold stress is diminished adaptive immunity. Immunohistochemistry and flow cytometry confirmed the presence of more CD8+ T cells (including antigen-specific cells) within the tumor microenvironment and spleens of animals at thermoneutral vs standard temperature. Conversely, animals at standard temperature had significantly increased infiltration of immunosuppressive cell subsets including GR-1+CD11b+ myeloid derived suppressor cells and Foxp3+ cells. These data reveal a fundamental relationship between energy utilized for thermoregulation and CD8+ T cell-dependent immunity and strongly suggest that immunological data collected from tumor bearing mice housed at standard temperature could be significantly biased by chronic cold stress.

During the course of experiments to determine the effects of elevated body temperature on anti-tumor immune responses, we unexpectedly observed that in control animals bearing tumors and maintained under standard room temperatures (IACUC-compliant, 21-23°C), the core temperature actually began to fall significantly as the tumors grew larger. Moreover, using thermal preference analyses, we found that animals bearing tumors exhibit considerable heat-seeking behavior. In an attempt to keep body temperature within the normal range (∼37°C), we began to house tumor bearing mice in a warmer environment (i.e., 28-30°C) considered to be the thermoneutral temperature for mice, as apposed to IACUC compliant temperatures which are considered mildly hypothermic for mice. Decisions that have resulted in the mildly cooler environmental temperature required in animal colonies have been generally based upon optimizing air-handling capacity of fumes and the thermal comfort of personnel rather than the optimal thermal comfort of animals (hence the requirements for multiple mice per cage to allow for huddling). When we analyzed tumor growth under thermoneutral conditions, syngeneic tumor growth was significantly delayed in animals maintained at 28-30°C compared to tumor growth in animals maintained at 21-23 oC. If the same comparison is made using SCID mice, no difference in tumor growth rate is observed. More specifically, BALB/c, C57BL/6 and SCID mice were housed in either standard room temperature (RT = 21-23°C) or in a warmer ambient temperature (WR = 28-30°C). Mice were then implanted with a syngeneic tumor (CT26 for BALB/c and SCID; B16.F10 for C57BL/6) subcutaneously and tumor growth was monitored. Tumor growth in C57BL/6 and BALB/c mice was inhibited when mice were housed in the warmer ambient temperature compared to animals maintained in standard room temperature. There was no difference in CT26 tumor growth in SCID mice. This finding suggested that the T cell-dependent anti-tumor immune response may be at least partly responsible for enhanced tumor growth control under conditions of warmer ambient temperature. Results of depletion studies in C57BL/6 mice bearing B16.F10 tumors, showed that both CD4+ and CD8+ T cells were required for inhibition of tumor growth in the warmer ambient temperature. In BALB/c mice bearing CT26 tumors, only CD8+ T cells were required to inhibit tumor growth. These findings suggest that the natural anti-tumor immune response exhibited by mice housed in standard colony conditions is sub-optimal and can be improved simply by housing mice under thermoneutral conditions. T cell dependent anti-tumor immunity appears to be at least one immune mechanism which is improved when animals are not cold stressed. Supported by NIH R01 CA 135368. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2011-790