Canine transmissible venereal tumour established in immunodeficient mice reprograms the gene expression profiles associated with a favourable tumour microenvironment to enable cancer malignancy.

BACKGROUND: Canine transmissible venereal tumours (CTVTs) can cross the major histocompatibility complex barrier to spread among dogs. In addition to the transmissibility within canids, CTVTs are also known as a suitable model for investigating the tumour-host immunity interaction because dogs live with humans and experience the same environmental risk factors for tumourigenesis. Moreover, outbred dogs are more appropriate than inbred mice models for simulating the diversity of human cancer development. This study built a new model of CTVTs, known as MCTVTs, to further probe the shaping effects of immune stress on tumour development. For xenotransplantation, CTVTs were first injected and developed in immunodeficient mice (NOD.CB17-Prkdcscid/NcrCrl), defined as XCTVTs. The XCTVTs harvested from NOD/SCID mice were then inoculated and grown in beagles and named mouse xenotransplantation of CTVTs (MCTVTs). RESULTS: After the inoculation of CTVTs and MCTVTs into immune-competent beagle dogs separately, MCTVTs grew faster and metastasized more frequently than CTVTs did. Gene expression profiles in CTVTs and MCTVTs were analysed by cDNA microarray to reveal that MCTVTs expressed many tumour-promoting genes involved in chronic inflammation, chemotaxis, extracellular space modification, NF-kappa B pathways, and focal adhesion. Furthermore, several well-known tumour-associated biomarkers which could predict tumour progression were overexpressed in MCTVTs. CONCLUSIONS: This study demonstrated that defective host immunity can result in gene instability and enable transcriptome reprogramming within tumour cells. Fast tumour growth in beagle dogs and overexpression of tumour-associated biomarkers were found in a CTVT strain previously established in immunodeficient mice. In addition, dysregulated interaction of chronic inflammation, chemotaxis, and extracellular space modification were revealed to imply the possibly exacerbating mechanisms in the microenvironments of these tumours. In summary, this study offers a potential method to facilitate tumour progression and provide a niche for discovering tumour-associated biomarkers in cancer research.

Xenograft cancer vaccines prepared from immunodeficient mice increase tumor antigen diversity and host T cell efficiency against colorectal cancers.

Autologous cancer vaccines (ACVs) are a desirable approach for personalized medicine, but the efficiency of ACVs remains unsatisfactory due to their low immunogenicity. This study developed a platform that can enhance the immunogenicity of ACVs by transplanting the tumors into immunodeficient mice. The CT26 cell line was inoculated into severe combined immunodeficient mice (SCID) for vaccine preparation where escalates tumor development, subsequently diversifying the tumor antigenic topology. CT26/SCID cancer vaccines significantly inhibited tumor growth, increased the amount of tumor infiltrating lymphocytes, and triggered Th-1 predominant immune responses. Tumor antigenic profiles of CT26/SCID cells were further analyzed by liquid chromatography-tandem mass spectrometry. Compared to CT26 parental cells, a total of 428 differentially expressed proteins (DEPs) were detected. These DEPs revealed that CT26/SCID cells overexpressed several novel therapeutic targets, including KNG1, apoA-I and, ß2-GPI, which can trigger cytotoxic T cells towards Th-1 predominant immune responses and directly suppress proliferation in tumors. CT26/SCID cancer vaccines can be easily manufactured, while traits of triggering stronger antigen-specific Th-1 immune activity against tumors, are retained. Results of this study provide an effective proof-of-concept of an ACV for personalized cancer immunotherapy.

Potential enhancement of host immunity and anti-tumor efficacy of nanoscale curcumin and resveratrol in colorectal cancers by modulated electro- hyperthermia.

BACKGROUND: Modulated electro-hyperthermia (mEHT) is a form of hyperthermia used in cancer treatment. mEHT has demonstrated the ability to activate host immunity by inducing the release of heat shock proteins, triggering apoptosis, and destroying the integrity of cell membranes to enhance cellular uptake of chemo-drugs in tumor cells. Both curcumin and resveratrol are phytochemicals that function as effective antioxidants, immune activators, and potential inhibitors of tumor development. However, poor bioavailability is a major obstacle for use in clinical cancer treatment. METHODS: This purpose of this study was to investigate whether mEHT can increase anti-cancer efficacy of nanosized curcumin and resveratrol in in vitro and in vivo models. The in vitro study included cell proliferation assay, cell cycle, and apoptosis analysis. Serum concentration was analyzed for the absorption of curcumin and resveratrol in SD rat model. The in vivo CT26/BALB/c animal tumor model was used for validating the safety, tumor growth curve, and immune cell infiltration within tumor tissues after combined mEHT/curcumin/resveratrol treatment. RESULTS: The results indicate co-treatment of mEHT with nano-curcumin and resveratrol significantly induced cell cycle arrest and apoptosis of CT26 cells. The serum concentrations of curcumin and resveratrol were significantly elevated when mEHT was applied. The combination also inhibited the growth of CT26 colon cancer by inducing apoptosis and HSP70 expression of tumor cells while recruiting CD3+ T-cells and F4/80+ macrophages. CONCLUSIONS: The results of this study have suggested that this natural, non-toxic compound can be an effective anti-tumor strategy for clinical cancer therapy. mEHT can enable cellular uptake of potential anti-tumor materials and create a favorable tumor microenvironment for an immunological chain reaction that improves the success of combined treatments of curcumin and resveratrol.

Relationship between Energy Dosage and Apoptotic Cell Death by Modulated Electro-Hyperthermia.

Modulated electro-hyperthermia (mEHT) is a form of mild hyperthermia (HT) used for cancer treatment. The principle utility of HT is the ability not only to increase cell temperature, but also to increase blood flow and associated pO2 to the microenvironment. While investigational evidence has shown the unique ability of mEHT to elicit apoptosis in cancer cells, in vivo and in vitro, the same trait has not been observed with conventional HT. There is dissension as to what allows mEHT to elicit apoptosis despite heating to only mild temperatures, with the predominant opinion in favor of increased temperature at a cellular level as the driving force. For this study, we hypothesized that in addition to temperature, the amount of electrical energy delivered is a major factor in induction of apoptosis by mEHT. To evaluate the impact of electrical energy on apoptosis, we divided generally practiced mEHT treatment into 3 phases: Phase I (treatment start to 10 min. mark): escalation from 25 °C to 37 °C Phase II (10 min. mark to 15 min. mark): escalation from 37 °C to 42 °C Phase III (15 min. mark to 45 min. mark): maintenance at 42 °C Combinations of mEHT at 18 W power, mEHT at 7.5 W power, water bath, and incubator were applied to each of the three phases. Power output was recorded per second and calculated as average power per second. Total number of corresponding Joules emitted per each experiment was also recorded. The biological effect of apoptotic cell death was assayed by annexin-V assay. In group where mEHT was applied for all three phases, apoptosis rate was measured at 31.18 ± 1.47%. In group where mEHT was only applied in Phases II and III, apoptosis rate dropped to 20.2 ± 2.1%. Where mEHT was only applied in Phase III, apoptosis was 6.4 ± 1.7%. Interestingly, when mEHT was applied in Phases I and II, whether Phase III was conducted in either water bath at 42 °C or incubator at 37 °C, resulted in nearly identical apoptosis rates, 26 ± 4.4% and 25.9 ± 3.1%, respectively. These results showed that accumulation of mEHT at high-powered setting (18 W/sec) during temperature escalation (Phase I and Phase II), significantly increased apoptosis of tested cancer cells. The data also showed that whereas apoptosis rate was significantly increased during temperature escalation by higher power (18 W/sec), apoptosis was limited during temperature maintenance with lower power (7.5 W/sec). This presents that neither maintenance of 42 °C nor accumulation of Joules by mEHT has immediate correlating effect on apoptosis rate. These findings may offer a basis for direction of clinical application of mEHT treatment.

Rapamycin and hydroxychloroquine combination alters macrophage polarization and sensitizes glioblastoma to immune checkpoint inhibitors.

INTRODUCTION: The failure of immune checkpoint inhibitor (ICPi) on glioblastoma (GBM) treatment underscores the need for improving therapeutic strategy. We aimed to change tumor associated macrophage (TAM) from M2 type (anti-inflammatory) to M1 (pro-inflammatory) type to increase the therapeutic response of ICPi. We proposed that combined rapamycin (R) and hydroxychloroquine (Q) preferentially induce M2 cells death, as fatty acid oxidation was their major source of energy. METHODS: Macrophage polarization was characterized on mice and human macrophage cell lines by specific cytokines stimulation with or without RQ treatment under single culture or co-culture with GBM cell lines. Tumor sizes were evaluated on subcutaneous and intracranial GL261 mice models with or without RQ, anti-PD1 mAb treatment. Tumor volumes assessed by MRI scan and proportions of tumor infiltrating immune cells analyzed by flow cytometry were compared. RESULTS: In vitro RQ treatment decreased the macrophages polarization of M2, increased the phagocytic ability, and increased the lipid droplets accumulation. RQ treatment decreased the expression levels of CD47 and SIRPα on tumor cells and macrophage cells in co-culture experiments. The combination of RQ and anti-PD1 treatment was synergistic in action. Enhanced the intra-tumoral M1/M2 ratio, the CD8/CD4 ratio in the intracranial GL261 tumor model after RQ treatment were evident. CONCLUSION: We provide a rationale for manipulating the macrophage phenotype and increased the therapeutic effect of ICPi. To re-educate and re-empower the TAM/microglia opens an interesting avenue for GBM treatment.

Signal Integration of IFN-I and IFN-II With TLR4 Involves Sequential Recruitment of STAT1-Complexes and NFκB to Enhance Pro-inflammatory Transcription.

Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (MΦ), and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)α, IFNγ, and Toll-like receptor (TLR)4 activate pro-inflammatory gene expression and are pro-atherogenic. Gene expression regulation of many pro-inflammatory genes has shown to rely on Signal Integration (SI) between IFNs and TLR4 through combinatorial actions of the Signal Transducer and Activator of Transcription (STAT)1 complexes ISGF3 and γ-activated factor (GAF), and Nuclear Factor-κB (NFκB). Thus, IFN pre-treatment ("priming") followed by LPS stimulation leads to enhanced transcriptional responses as compared to the individual stimuli. To characterize the mechanism of priming-induced IFNα + LPS- and IFNγ + LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, MΦ, and DC in response to IFNα, IFNγ, and/or LPS. Thus, we identified IFNα + LPS or IFNγ + LPS induced genes commonly expressed in these cell types that bound STAT1 and p65 at comparable γ-activated sequence (GAS), Interferon-stimulated response element (ISRE), or NFκB sites in promoter proximal and distal regions. Comparison of the relatively high number of overlapping ISRE sites in these genes unraveled a novel role of ISGF3 and possibly STAT1/IRF9 in IFNγ responses. In addition, similar STAT1-p65 co-binding modes were detected for IFNα + LPS and IFNγ + LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NFκB or ISRE/NFκB composite sites already upon IFNα or IFNγ treatment. This STAT1-p65 co-binding significantly increased after subsequent LPS exposure and correlated with histone acetylation, PolII recruitment, and amplified target gene transcription in a STAT1-p65 co-bound dependent manner. Thus, co-binding of STAT1-containing transcription factor complexes and NFκB, activated by IFN-I or IFN-II together with LPS, provides a platform for robust transcriptional activation of pro-inflammatory genes. Moreover, our data offer an explanation for the comparable effects of IFNα or IFNγ priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.

Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells.

PURPOSE: Modulated electro-hyperthermia (mEHT) stands to be a significant technological advancement in the hyperthermia field, utilizing autofocusing electromagnetic power on the cell membrane to create massive apoptosis. Since mEHT possesses the unique ability to excite cell membranes, we hypothesized that mEHT could enhance the uptake of liposomal drugs by enhancing phagocytic activity. MATERIALS AND METHODS: Water bath control and mEHT were used to compare the enhancement of liposome-encapsulated doxorubicin (Lipodox®) uptake by cancer cells. Cancer cells were made visible by doxorubicin fluorescence to investigate drug uptake. Viable cell yield was determined via the Trypan Blue exclusion method. Various substrates were used to investigate the mechanism of drug-uptake enhancement. The murine colon carcinoma model, CT26, was used to confirm the tissue infiltration of Lipodox® and its therapeutic effect. RESULTS: mEHT treatment showed a significant enhancement of Lipodox® uptake of doxorubicin fluorescence compared with 37°C or 42°C water bath treatment. Tumor tissue sections also confirmed that mEHT treatment achieved the highest doxorubicin concentration in vivo (1.44±0.32 µg/g in mEHT group and 0.79±0.32 µg/g in 42°C water bath). Wortmannin was used to inhibit the macropinocytosis effect and 70 kDa dextran-FITC served as uptake substance. The uptake of dextran-FITC by cancer cells significantly increased after mEHT treatment whereas such enhancement was significantly inhibited by wortmannin. CONCLUSION: The result showed mEHT-induced particle-uptake through macropinocytosis. mEHT-enhanced uptake of Lipodox® may amplify the therapeutic effect of liposomal drugs. This novel finding warrants further clinical investigation.

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

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

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

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

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

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

Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy.

BACKGROUND: The treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs. METHODS: The CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied. RESULTS: mEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity. CONCLUSIONS: This study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy.

Gene-expression profiling to identify genes related to spontaneous tumor regression in a canine cancer model.

Microarray transcriptome study in cancer has been commonly used to investigate tumorigenic mechanisms. The unique growth pattern of spontaneous regression (SR) after progressive (P) growth in canine transmissible venereal tumor (CTVT) provides a valuable cancer model to study the genome-wide differences in samples between the two stages of growth. In this study, Affymetrix analysis was performed based on the canine genome to compare the gene expression profiles of CTVT P- and SR-phase tumors. A total of 459 (278 up-regulated and 181 down-regulated) genes were identified as being differentially-expressed during the SR phase by the 2-fold method. Further analysis of these genes revealed that the expression of three genes associated with IL-6 production -TIMD-4, GPNMB and PLTP - was significantly higher in SR-phase tumors than in P-phase tumors; these results were also confirmed by real time RT-PCR in tumor tissues of beagles. In addition, we found that Th17-related genes were over-expressed in the SR phase, suggesting autoimmune responses involvement in tumor regression. Although the interaction between CTVT and host immunity were partially investigated in previous studies, our results enable us to gain new insight into the genes and possible mechanisms involved in tumor regression and reveal potentially useful targets for cancer therapy.

Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor.

BACKGROUND: Chemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-ß derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-ß in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT). RESULTS: We have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1ß and TGF-ß had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression. CONCLUSION: CXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.

Forty-photon-per-pulse spectral phase retrieval by shaper-assisted modified interferometric field autocorrelation.

We retrieve the spectral phase of 400 fs pulses at 1560 nm with 5.2 aJ coupled pulse energy (40 photons) by the modified interferometric field autocorrelation method, using a pulse shaper and a 5 cm long periodically poled lithium niobate waveguide. The carrier-envelope phase control of the shaper can reduce the fringe density of the interferometric trace and permits longer lock-in time constants, achieving a sensitivity of 2.7×10(-9) mW(2) (40 times better than the previous record for self-referenced nonlinear pulse measurement). The high stability of the pulse shaper allows for accurate and reproducible measurements of complicated spectral phases.

Transient downregulation of monocyte-derived dendritic-cell differentiation, function, and survival during tumoral progression and regression in an in vivo canine model of transmissible venereal tumor.

Tumors often target dendritic cells (DCs) to evade host immune surveillance. DC injury is reported in many rodent and human tumors but seldom in tumors of other mammals. Canine transmissible venereal tumor (CTVT), a unique and spontaneous cancer transmitted by means of viable tumor cells. CTVT causes manifold damage to monocyte-derived DCs. This cancer provides an in vivo model of cancer to study the role of monocyte-derived DCs during spontaneous regression. Using flow cytometry and real-time reverse-transcription polymerase chain reactions, we compared the expression of surface molecules on monocyte-derived DCs between normal dogs and dogs with CTVT. These markers were CD1a, CD83, costimulatory factors (CD40, CD80, and CD86), and major histocompatability complex classes I and II. In immature DCs (iDCs) and lipopolysaccharide-treated mature DCs (mDCs), the surface markers were mostly downregulated during tumoral progression and regression. The tumor lowered endocytic activity of iDCs, as reflected in dextran uptake, and decreased allogeneic mixed lymphocyte reactions of mDCs. In addition, it decreased the number of monocytes in the peripheral blood by 40%. The tumor substantially impaired the efficiency with which DCs were generated from monocytes and with which mDCs were generated from iDCs. We also found that progression-phase CTVT supernatants that were cultured for 48 h and that contained protein components killed both monocytes and DCs. Additionally, DC numbers were significantly lower in the draining lymph nodes in CTVT dogs than in normal dogs. In conclusion, CTVT caused devastating damage to monocyte-derived DCs; this might be one of its mechanisms for evading host immunity. Reestablishment of monocyte-derived DC activity by the host potentially might contribute to spontaneous tumoral regression. These findings provide insight into the extent of tumoral effects on host immune systems and responses. This information is useful for developing cancer immunotherapies.