Hui-Yen Chuang

Antitumor effect of orlistat, a fatty acid synthase inhibitor, is via activation of caspase-3 on human colorectal carcinoma-bearing animal.

We established a HT-29/tk-luc human colorectal carcinoma-bearing animal model for the study of the inhibition effect and mechanism of orlistat, a fatty acid synthase (FASN) inhibitor. The results showed that orlistat caused cell cycle arrest at G1 phase, and triggered apoptosis through caspase-3 activation. The tumor inhibition effect of orlistat may also due to the inhibition of fatty acid synthesis without altering FASN activity. The tumor size of orlistat-treated mice in vivo was significantly smaller than that of the controls with 55% inhibition. The therapeutic efficacy was further confirmed with the bioluminescent imaging and nuclear molecular imaging with ¹³¹I-FIAU/gamma scintigraphy and ¹¹C-acetate/microPET. We suggest that FASN is a potential target for the treatment of human colorectal carcinoma.

Targeted therapy with fatty acid synthase inhibitors in a human prostate carcinoma LNCaP/ tk-luc -bearing animal model

Background:Fatty acid synthase (FASN) is highly upregulated in human prostate carcinomas. Inhibition of FASN could arrest cell cycle and trigger apoptosis rapidly, implying the reliance of cancer cell survival on FASN. However, little is known about the effect of C75, a FASN inhibitor, and siFASN (that is, small interfering RNA targeted at FASN) on prostate cancer in living subjects.Methods:We used C75 and siFASN to mediate the endogenous fatty acid metabolism in LNCaP human prostate cancer cells stably expressing herpes simplex virus type 1 thymidine kinase (HSV1-tk) and luciferase (luc) reporter genes, and assessed the effect of FASN blockade with different schedules of administration on tumor growth using noninvasive molecular imaging.Results:FASN blockade exhibited the proliferative inhibition and induced G1-phase cell cycle arrest of LNCaP cells. For in vivo studies, the tumor growth inhibition by C75 (total 120 mg kg−1; 30 mg kg−1 once a week or 15 mg kg−1 twice a week for 4 weeks) and siFASN (1.4 mg kg−1 every alternate day up to 16 days) treatments were 80% and 70%, respectively, compared with that of the control.Conclusion:The results suggest that C75 may be superior to siFASN in anticancer effect on prostate cancer.

Immunomodulation of Curcumin on Adoptive Therapy with T Cell Functional Imaging in Mice

Adoptive T-cell therapy involves the ex vivo expansion and subsequent transfusion of tumor-specific T lymphocytes to eliminate tumors. Using immune modulators to block immunosuppressive factors in the tumor microenvironment has emerged as a promising strategy to enhance T-cell–mediated tumor regression. Curcumin, a major component of turmeric, has been shown to possess antitumor and immunomodulatory effects by regulating a diverse range of molecular targets. Thus, we hypothesize that these beneficial effects of curcumin may improve the therapeutic efficacy of adoptive therapy. Here, we have shown that curcumin enhances cytotoxicity of CD8+ T cells toward tumors via alteration of the tumor microenvironment when combined with adoptive therapy. We found that T-cell accumulation and function were increased in combined treatment due to the blockade of different immunosuppressors, including TGF-β, indoleamine 2,3-dioxygenase, and regulatory T cells. Furthermore, bioluminescent imaging with a granzyme B promoter–conjugated optical reporter also reflected improved cytotoxicity of antigen-specific CD8+ T cells in tumor-bearing mice during treatment. These findings suggest that combination of multitargeting drugs, such as curcumin, with adoptive therapy may have potential for clinical application. In addition, using a granzyme B–specific imaging reporter to assess T-cell function may also be applied for the development and therapeutic evaluation of new immunotherapy in preclinical studies. Cancer Prev Res; 5(3); 444–52. ©2011 AACR.

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

Significance Blood-based cancer diagnosis is highly attractive, but current strategies suffer because they rely on the detection of endogenous molecules that often are secreted into the circulation by both malignant and nonmalignant cells. One solution to this problem is to avoid nonmalignant tissue expression by artificially engineering tumor cells to express a unique reporter not normally expressed by any tissue. This study shows that systemic administration of nonviral safe vectors we call “tumor-activatable minicircles” allows one to distinguish tumor-bearing from tumor-free subjects reliably and to assess tumor burden simply by measuring blood levels of such a reporter. Our system represents an alternative paradigm for improved cancer detection and could enable more timely interventions to combat this devastating disease. 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 (r2 = 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.

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.

Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×106 cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cells genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

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-1β, 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-1β, (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.

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 Burkitts 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.

Abstract 1099: Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Many chemotherapeutic agents are known to have immunomodulatory activity to improve the effectiveness of adoptive T-cell therapy. We hypothesized that sorafenib could reverse the immunosuppression of tumor microenvironment to enhance T-cell mediated tumor regression. Experimental design: OVA-specific CD8+ T cells were co-cultured with sorafenib-pretreated OVA-overexpressing E.G7 mouse lymphoma cells to evaluate their function using flow cytometry, 51Cr-release assay, and transwell assay. The underlying mechanism of sorafenib-mediated immunomodulation was also explored by Western blotting. In animal studies, the activation of OVA-specific CD8+ T cells transduced with a granzyme B promoter-driven imaging reporter was monitored by bioluminescence imaging and the levels of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were assessed by flow cytometry. Results: We found that sorafenib suppressed the expression of critical immunosuppressive components, including TGF-β , IL-10, VEGF, indoleamine 2,3-dioxygenase (IDO) and CCL2/MCP-1, in E.G7 cells through the inhibition of STAT3 signaling pathway and decreased the percentage of Tregs and MDSCs in tumors. Surprisingly, the anti-tumor effect of small numbers of CD8+ T cells was substantially enhanced when combined with sorafenib in vivo. Citation Format: Jeng-Jong Hwang, Hui-Yen Chuang, Ya-Fang Chang. Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment. [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 1099. doi:10.1158/1538-7445.AM2014-1099