Orlistat Resensitizes Sorafenib-Resistance in Hepatocellular Carcinoma Cells through Modulating Metabolism.

Sorafenib is one of the options for advanced hepatocellular carcinoma treatment and has been shown to extend median overall survival. However, sorafenib resistance often develops a few months after treatment. Hence, developing various strategies to overcome sorafenib resistance and understand the possible mechanisms is urgently needed. We first established sorafenib-resistant hepatocellular carcinoma (HCC) cells. Then, we found that sorafenib-resistant Huh7 cells (Huh7/SR) exhibit higher glucose uptakes and express elevated fatty acid synthesis and glucose metabolism-related proteins than their parental counterparts (Huh7). The current study investigated whether sorafenib resistance could be reversed by suppressing fatty acid synthesis, using a fatty acid synthase (FASN) inhibitor, orlistat, in HCC cells. FASN inhibition-caused changes in protein expressions and cell cycle distribution were analyzed by Western blot and flow cytometry, and changes in glucose uptakes were also evaluated by 18F-FDG uptake. Orlistat remarkably enhanced the cytotoxicity of sorafenib in both Huh7 and Huh7/SR cells, and flow cytometry showed that combination treatment significantly increased the sub-G1 population in both cell lines. Western blot revealed that the combination treatment effectively increased the ratio of Bax/Bcl-2 and decreased expressions of pERK; additionally, the combination treatment also strongly suppressed fatty acid synthesis-related proteins (e.g., FASN and SCD) in both cell lines. Lastly, the 18F-FDG uptake was repressed by the combination treatment in both cell lines. Our results indicated that orlistat-mediated FASN inhibition could overcome sorafenib resistance and enhance cell killing in HCC by changing cell metabolism.

Detecting cancers through tumor-activatable minicircles that lead to a detectable blood biomarker.

Earlier detection of cancers can dramatically improve the efficacy of available treatment strategies. However, despite decades of effort on blood-based biomarker cancer detection, many promising endogenous biomarkers have failed clinically because of intractable problems such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. In this work we present a tumor-detection strategy based on systemic administration of tumor-activatable minicircles that use the pan-tumor-specific Survivin promoter to drive expression of a secretable reporter that is detectable in the blood nearly exclusively in tumor-bearing subjects. After systemic administration we demonstrate a robust ability to differentiate mice bearing human melanoma metastases from tumor-free subjects for up to 2 wk simply by measuring blood reporter levels. Cumulative change in reporter levels also identified tumor-bearing subjects, and a receiver operator-characteristic curve analysis highlighted this test's performance with an area of 0.918 ± 0.084. Lung tumor burden additionally correlated (r(2) = 0.714; P < 0.05) with cumulative reporter levels, indicating that determination of disease extent was possible. Continued development of our system could improve tumor detectability dramatically because of the temporally controlled, high reporter expression in tumors and nearly zero background from healthy tissues. Our strategy's highly modular nature also allows it to be iteratively optimized over time to improve the test's sensitivity and specificity. We envision this system could be used first in patients at high risk for tumor recurrence, followed by screening high-risk populations before tumor diagnosis, and, if proven safe and effective, eventually may have potential as a powerful cancer-screening tool for the general population.

Longitudinal Monitoring of Antibody Responses against Tumor Cells Using Magneto-nanosensors with a Nanoliter of Blood.

Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.

Curcumin Enhances the Abscopal Effect in Mice with Colorectal Cancer by Acting as an Immunomodulator.

Radiotherapy (RT) is an effective cancer treatment. The abscopal effect, referring to the unexpected shrinkage observed in non-irradiated tumors after radiation therapy, is thought to be mediated by systemic immune activation. However, it has low incidence and is unpredictable. Here, RT was combined with curcumin to investigate how curcumin affects RT-induced abscopal effects in mice with bilateral CT26 colorectal tumors. Indium 111-labeled DOTA-anti-OX40 mAb was synthesized to detect the activated T cell accumulations in primary and secondary tumors correlating with the changes in protein expressions and tumor growth to understand the overall effects of the combination of RT and curcumin. The combination treatment caused the most significant tumor suppression in both primary and secondary tumors, accompanied by the highest 111In-DOTA-OX40 mAb tumor accumulations. The combination treatment elevated expressions of proapoptotic proteins (Bax and cleaved caspase-3) and proinflammatory proteins (granzyme B, IL-6, and IL-1ß) in both primary and secondary tumors. Based on the biodistribution of 111In-DOTA-OX40 mAb, tumor growth inhibition, and anti-tumor protein expression, our findings suggest that curcumin could act as an immune booster to augment RT-induced anti-tumor and abscopal effects effectively.

Cancer Detection Using an Artificial Secretable MicroRNA Found in Blood and Urine.

Biomarkers can potentially help in the detection and prognosis of diseases such as cancer, its recurrence, predicting response to therapy, and monitoring of response during and/or after treatment. Endogenous tumor blood biomarkers suffer from low concentrations that are not distinguishable from background noise and, if identified, the localization of the biomarker production site is not known. The use of exogenously introduced or artificial biomarkers can eliminate these issues. In this study, we show that cancer cells can be made to produce an artificial secreted microRNA (Sec-miR) that can be detected in media from cells in culture, and from both blood and urine in living mice. In culture, we show that chaining a number of Sec-miR sequences in a plasmid and transfecting cells with the plasmids could increase Sec-miR secretion as the number of sequences increases. Tumor induction in mice with a stably transfected HeLa cell line shows the presence and significant increase in the Sec-miR with time and tumor growth in plasma (p < 0.001, R2 = 0.5542). The relative half-life of the Sec-miR was seen to be 1.2 h in the plasma of living mice and was seen to appear in urine within 12 h. The transgene for the Sec-miR within a minicircle was introduced via the tail-vein into subcutaneous tumor-bearing mice. As the tumor growth increased with time, further in vivo transfection of the Sec-miR minicircles showed an increase in Sec-miR in both plasma and urine (R2 = 0.4546). This study demonstrated that an exogenous Sec-miR biomarker would allow for early tumor detection using in vitro diagnostics techniques.

Liposomal ß-Sitosterol Suppresses Metastasis of CT26/luc Colon Carcinoma via Inhibition of MMP-9 and Evoke of Immune System.

ß-sitosterol (SITO) has been reported with anticancer effects; however, with poor bioavailability. The current study aimed to investigate whether liposomal encapsulated ß-sitosterol (LS) has a better inhibition effect on tumor metastasis than ß-sitosterol in a CT26/luc lung metastasis mouse model and the possible underlying mechanism. LS was liposomal-encapsulated SITO and was delivered to mice by oral gavage. The cell viability was determined by the MTT assay, and invasiveness of the tumor cells and related protein expression were evaluated with the invasion assay and Western blotting. For therapeutic efficacy evaluation, male BALB/c mice were treated with PBS, SITO, and LS once a day for 7 days prior to intravenous injections of CT26/luc cells; treatments were continued twice a week post-cell inoculation throughout the entire experiment. Tumor growth inhibition was monitored by bioluminescent imaging (BLI). IL-12, IL-18, and IFN-γ in the intestinal epithelium were determined by ELISA. The results show that LS treatment had a better invasion inhibition with lower cytotoxicity than SITO when the same dose was utilized. Notably, mice treated with LS significantly exhibited fewer metastases to the lungs and other tissues/organs compared with the Control and SITO groups. Additionally, the IL-12, IL-18, and IFN-γ levels were significantly increased in the LS-treated mice compared with the Control and SITO groups. The underlying mechanism may be through the inhibition of MMP-9 and elicitation of the antitumoral Th1 immune response, such as increasing CD4+ and CD8+ T cells, IL-12, IL-18, and IFN-γ.

Effects of orlistat combined with enzalutamide and castration through inhibition of fatty acid synthase in a PC3 tumor-bearing mouse model.

Androgen deprivation therapy (ADT) is one of the typical treatments used for patients with prostate cancer (PCa). ADT, however, may fail when PCa develops castration-resistance. Fatty acid synthase (FASN), a critical enzyme involved in fatty acid synthesis, is found to be up-regulated in PCa. Since enzalutamide and ADT are frequently used for the treatment of PCa, the present study aimed to unravel the underlying mechanism of combination of orlistat, an FASN inhibitor, and enzalutamide using PC3 cell line; and orlistat and castration in PC3 tumor-bearing animal model. Cytotoxicity was determined by AlamarBlue assay. Drug effects on the cell cycle and protein expressions were assayed by the flow cytometry and Western blot. Electromobility shift assay was used to evaluate the NF-κB activity. The tumor growth delay, expressions of the signaling-related proteins, and histopathology post treatments of orlistat and castration were evaluated in PC3 tumor-bearing mouse model. The results showed that orlistat arrested the PC3 cells at the G1 phase of the cell cycle and enhanced the cytotoxic effects of enzalutamide synergistically. Pretreatment with orlistat combined with castration inhibited the tumor growth significantly compared with those of castration and orlistat treatments alone in PC3 tumor-bearing mice. Combination treatment reduced both FASN and NF-κB activities and their downstream effector proteins. The present study demonstrated the synergistic effects of orlistat combined with enzalutamide in vitro and castration in vivo on human PCa.

A combination of sorafenib and radiotherapy reduces NF-κB activity and growth of hepatocellular carcinoma in an orthotopic mouse model.

Hepatocellular carcinoma (HCC) is difficult to diagnose at an early stage, and its prognosis is generally poor. Sorafenib is the primary treatment for unresectable advanced HCC and targets multiple receptor tyrosine kinases. However, sorafenib only extends the average survival time by 3 months. This observation indicates that sorafenib may need to be combined with other treatments to further improve outcomes. We previously showed that combination of sorafenib with radiotherapy (RT) enhances tumor inhibition in subcutaneous HCC mouse models compared with monotherapy. The present study demonstrated that combining sorafenib and RT could suppress tumor growth in an orthotopic HCC model by regulating apoptosis and NF-κB-related pathways. Moreover, decreased numbers of visible liver tumors and a smaller percentage of spleen metastases were found in the combination group. A transient drop in body weight was initially observed after RT, but progressive recovery of body weight occurred. The current study showed that the combination of sorafenib and RT could be a safe strategy for HCC treatment.

Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy.

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

The Association of MMP7 Promoter Polymorphisms With Gastric Cancer.

BACKGROUND/AIM: Few studies have examined the genetic role of matrix metalloproteinases (MMPs) to early detection or prediction in gastric cancer development. In this study, the contribution of MMP7 promoter (A-181G and C-153T) polymorphic genotypes to gastric cancer risk in Taiwanese was investigated for the first time. MATERIALS AND METHODS: A total of 121 cases and 363 controls were enrolled and their MMP7 genotypes at A-181G and C-153T were examined by polymerase chain reaction-restriction fragment length polymorphism methodology using genomic DNA from serum. RESULTS: The GG genotype at MMP7 A-181G was found to represent a risk factor for gastric cancer, especially among smokers. No individual with variant genotype carrier at MMP7 C-153T was found among this Taiwanese population. CONCLUSION: The G allele of MMP7 A-181G may serve as an early predictor for gastric cancer risk in Taiwanese; other gastric cancer markers are still urgently needed.

Fatty Acid Inhibition Sensitizes Androgen-Dependent and -Independent Prostate Cancer to Radiotherapy via FASN/NF-κB Pathway.

Elevated fatty acid synthase (FASN) has been reported in both androgen-dependent and -independent prostate cancers. Conventional treatment for prostate cancer is radiotherapy (RT); however, the following radiation-induced radioresistance often causes treatment failure. Upstream proteins of FASN such as Akt and NF-κB are found increased in the radioresistant prostate cancer cells. Nevertheless, whether inhibition of FASN could improve RT outcomes and reverse radiosensitivity of prostate cancer cells is still unknown. Here, we hypothesised that orlistat, a FASN inhibitor, could improve RT outcomes in prostate cancer. Orlistat treatment significantly reduced the S phase population in both androgen-dependent and -independent prostate cancer cells. Combination of orlistat and RT significantly decreased NF-κB activity and related downstream proteins in both prostate cancer cells. Combination effect of orlistat and RT was further investigated in both LNCaP and PC3 tumour-bearing mice. Combination treatment showed the best tumour inhibition compared to that of orlistat alone or RT alone. These results suggest that prostate cancer treated by conventional RT could be improved by orlistat via inhibition of FASN.

Enhanced cytotoxicity of human hepatocellular carcinoma cells following pretreatment with sorafenib combined with trichostatin A.

Trichostatin A (TSA), a hydroxamate histone deacetylase inhibitor, is a compound that has been identified to induce anticancer activity. The aim of the present study was to investigate whether sorafenib, in combination with TSA, was able to augment the anticancer effects of TSA, identifying an optimum treatment time plan and the potential underlying molecular mechanisms involved in human hepatocellular carcinoma (HCC) in vitro. Huh7/nuclear factor-κB (NF-κB)-luc2 cells were treated with TSA or sorafenib alone, or sorafenib, prior to, in combination with or following TSA treatment. Huh7/NF-κB-luc2 cell viability following TSA treatment was determined using an MTT assay, and NF-κB activity was analyzed. In addition, the expression levels of NF-κB-regulated downstream effector proteins were assayed by western blotting. Inhibitors of mitogen-activated protein kinases (MAPKs), protein kinase B (AKT) and mutant inhibitor of NF-κBα (IκBαM) vectors were used to confirm the function of the NF-κB signal transduction pathways in response to the effects of sorafenib combined with TSA against HCC. The results of the present study indicated that pre-treatment with sorafenib followed by TSA inhibited the cell viability compared with other treatment modalities, and prevented TSA-induced extracellular-signal-regulated kinase (ERK)/NF-κB activity and expression of downstream effector proteins. It was further demonstrated that IκBαM vector sensitized Huh7/NF-κB-luc2 cells to TSA, thus it was possible to reverse TSA-induced NF-κB activity using PD98059, a MAPK/ERK kinase inhibitor. In conclusion, sorafenib pre-treatment may increase the efficacy of subsequent TSA treatment in HCC. Furthermore, sorafenib pre-treatment is hypothesized to sensitize HCC to TSA via the inhibition of the MEK/ERK/NF-κB signal transduction pathway.

Engineered immune cells as highly sensitive cancer diagnostics.

Endogenous biomarkers remain at the forefront of early disease detection efforts, but many lack the sensitivities and specificities necessary to influence disease management. Here, we describe a cell-based in vivo sensor for highly sensitive early cancer detection. We engineer macrophages to produce a synthetic reporter on adopting an M2 tumor-associated metabolic profile by coupling luciferase expression to activation of the arginase-1 promoter. After adoptive transfer in colorectal and breast mouse tumor models, the engineered macrophages migrated to the tumors and activated arginase-1 so that they could be detected by bioluminescence imaging and luciferase measured in the blood. The macrophage sensor detected tumors as small as 25-50 mm3 by blood luciferase measurements, even in the presence of concomitant inflammation, and was more sensitive than clinically used protein and nucleic acid cancer biomarkers. Macrophage sensors also effectively tracked the immunological response in muscle and lung models of inflammation, suggesting the potential utility of this approach in disease states other than cancer.

A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

Lupeol acetate ameliorates collagen-induced arthritis and osteoclastogenesis of mice through improvement of microenvironment.

Lupeol has been shown with anti-inflammation and antitumor capability, however, the poor bioavailability limiting its applications in living subjects. Lupeol acetate (LA), a derivative of lupeol, shows similar biological activities as lupeol but with better bioavailability. Here RAW 264.7 cells and bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS) were treated with 0-80µM of LA, and assayed for TNF-α, IL-1ß, COX-2, MCP-1 using Western blotting. Moreover, osteoclatogenesis was examined with reverse transcription PCR (RT-PCR) and tartrate-resistant acid phosphatase (TRAP) staining. For in vivo study, collagen-induced arthritis (CIA)-bearing DBA/1J mice were randomly separated into three groups: vehicle, LA-treated (50mg/kg) and curcumin-treated (100mg/kg). Therapeutic efficacies were assayed by the clinical score, expression levels of serum cytokines including TNF-α and IL-1ß, (18)F-fluorodeoxyglucose ((18)F-FDG) microPET/CT and histopathology. The results showed that LA could inhibit the activation, migration, and formation of osteoclastogenesis of macrophages in a dose-dependent manner. In RA-bearing mice, the expressions of inflammation-related cytokines were suppressed, and clinical symptoms and bone erosion were ameliorated by LA. The accumulation of (18)F-FDG in the joints of RA-bearing mice was also significantly decreased by LA. The results indicate that LA significantly improves the symptoms of RA by down-regulating expressions of inflammatory cytokines and osteoclastogenesis.

Artificial MicroRNAs as Novel Secreted Reporters for Cell Monitoring in Living Subjects.

Reporter genes are powerful technologies that can be used to directly inform on the fate of transplanted cells in living subjects. Imaging reporter genes are often employed to quantify cell number, location(s), and viability with various imaging modalities. To complement this, reporters that are secreted from cells can provide a low-cost, in vitro diagnostic test to monitor overall cell viability at relatively high frequency without knowing the locations of all cells. Whereas protein-based secretable reporters have been developed, an RNA-based reporter detectable with amplification inherent PCR-based assays has not been previously described. MicroRNAs (miRNAs) are short non-coding RNAs (18-22 nt) that regulate mRNA translation and are being explored as relatively stable blood-based disease biomarkers. We developed an artificial miRNA-based secreted reporter, called Sec-miR, utilizing a coding sequence that is not expressed endogenously and does not have any known vertebrate target. Sec-miR was detectable in both the cells and culture media of transiently transfected cells. Cells stably expressing Sec-miR also reliably secreted it into the culture media. Mice implanted with parental HeLa cells or HeLa cells expressing both Sec-miR and the bioluminescence imaging (BLI) reporter gene Firefly luciferase (FLuc) were monitored over time for tumor volume, FLuc signal via BLI, and blood levels of Sec-miR. Significantly (p<0.05) higher Sec-miR was found in the blood of mice bearing Sec-miR-expressing tumors compared to parental cell tumors at 21 and 28 days after implantation. Importantly, blood Sec-miR reporter levels after day 21 showed a trend towards correlation with tumor volume (R2 = 0.6090; p = 0.0671) and significantly correlated with FLuc signal (R2 = 0.7067; p<0.05). Finally, we could significantly (p<0.01) amplify Sec-miR secretion into the cell media by chaining together multiple Sec-miR copies (4 instead of 1 or 2) within an expression cassette. Overall, we show that a novel complement of BLI together with a unique Sec-miR reporter adds an in vitro RNA-based diagnostic to enhance the monitoring of transplanted cells. While Sec-miR was not as sensitive as BLI for monitoring cell number, it may be more sensitive than clinically-relevant positron emission tomography (PET) reporter assays. Future work will focus on improving cell detectability via improved secretion of Sec-miR reporters from cells and more sensitive detection platforms, as well as, exploring other miRNA sequences to allow multiplexed monitoring of more than one cell population at a time. Continued development may lead to more refined and precise monitoring of cell-based therapies.

Sorafenib pretreatment enhances radiotherapy through targeting MEK/ERK/NF-κB pathway in human hepatocellular carcinoma-bearing mouse model.

Patients with unresectable hepatocellular carcinoma (HCC) usually have poor prognosis because current monotherapy including surgery, chemotherapy and radiotherapy (RT) are not effective. Combination therapy may be effective to overcome this clinical problem. Here, we proposed the combination of sorafenib and RT, which have been applied in HCC treatment, could improve the treatment outcome of HCC. Our previous study showed that sorafenib could suppress the expression of NF-κB which is related to the chemo- and radio-resistance. Nevertheless, the expression of NF-κB is oscillatory and is affected by the treatments. Thus, understanding the oscillation of NF-κB expression would be beneficial for determining the optimal treatment schedule in combination therapy. Here established Huh7/NF-κB-tk-luc2/rfp cell line, in which NF-κB indicates a NF-κB promoter, was utilized to noninvasively monitor the expression of NF-κB overtime in vitro and in vivo. The results show that pretreatment of sorafenib with RT suppresses the expressions of NF-κB and its downstream proteins induced by radiation through downregulation of phosphorylated extracellular signal-regulated kinase (pERK) most significantly compared with other treatment schedules. The results were further verified with Western blotting, EMSA, and NF-κB molecular imaging. These findings suggest that pretreatment of sorafenib with RT may be the ideal treatment schedule for the treatment of HCC.

CD44 and CD24 coordinate the reprogramming of nasopharyngeal carcinoma cells towards a cancer stem cell phenotype through STAT3 activation.

Cell surface proteins such as CD44 and CD24 are used to distinguish cancer stem cells (CSCs) from the bulk-tumor population. However, the molecular functionalities of CD24 and CD44, and how these two molecules coordinate in CSCs remain poorly understood. We found that nasopharyngeal carcinoma (NPC) cells with high expression of CD44 and CD24 proteins presented with pronounced CSC properties. Accordingly, a subpopulation of NPC cells with co-expression of CD44 and CD24 were specially enriched in high-stage clinical samples. Furthermore, ectopically expressing the epithelial-mesenchymal transition (EMT) regulator Twist was able to upregulate the stemness factors, and vice versa. This indicates a reciprocal regulation of stemness and EMT. Intriguingly, we found that this reciprocal regulation was differentially orchestrated by CD44 and CD24, and only simultaneous silencing the expression of CD44 and CD24 led to a broad-spectrum suppression of CSC properties. Oppositely, overexpression of CD44 and CD24 induced the reprogramming of parental NPC cells into CSCs through STAT3 activation, which could be blunted by STAT3 inhibition, indicating that CD44 and CD24 collaboratively drive the reprogramming of NPC cells through STAT3-mediated stemness and EMT activation. Consequently, targeting of the CD44/CD24/STAT3 axis may provide a potential therapeutic paradigm for the treatment of NPC through repressing CSC activities.

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice.

Ex vivo expansion of CD8+ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8+ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8+ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8+ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2x106 CD8+ T-cells and achieved the same level of tumor growth inhibition as that of 5x106 CD8+ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-ß, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8+ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8+ T-cells which also stay longer.

Serial low doses of sorafenib enhance therapeutic efficacy of adoptive T cell therapy in a murine model by improving tumor microenvironment.

Requirements of large numbers of transferred T cells and various immunosuppressive factors and cells in the tumor microenvironment limit the applications of adoptive T cells therapy (ACT) in clinic. Accumulating evidences show that chemotherapeutic drugs could act as immune supportive instead of immunosuppressive agents when proper dosage is used, and combined with immunotherapy often results in better treatment outcomes than monotherapy. Controversial immunomodulation effects of sorafenib, a multi-kinases inhibitor, at high and low doses have been reported in several types of cancer. However, what is the range of the low-dose sorafenib will influence the host immunity and responses of ACT is still ambiguous. Here we used a well-established E.G7/OT-1 murine model to understand the effects of serial low doses of sorafenib on both tumor microenvironment and transferred CD8+ T cells and the underlying mechanisms. Sorafenib lowered the expressions of immunosuppressive factors, and enhanced functions and migrations of transferred CD8+ T cells through inhibition of STAT3 and other immunosuppressive factors. CD8+ T cells were transduced with granzyme B promoter for driving imaging reporters to visualize the activation and distribution of transferred CD8+ T cells prior to adoptive transfer. Better activations of CD8+ T cells and tumor inhibitions were found in the combinational group compared with CD8+ T cells or sorafenib alone groups. Not only immunosuppressive factors but myeloid derived suppressive cells (MDSCs) and regulatory T cells (Tregs) were decreased in sorafenib-treated group, indicating that augmentation of tumor inhibition and function of CD8+ T cells by serial low doses of sorafenib were via reversing the immunosuppressive microenvironment. These results revealed that the tumor inhibitions of sorafenib not only through eradicating tumor cells but modifying tumor microenvironment, which helps outcomes of ACT significantly.