Treatment Of Tumor-bearing Mice Research Articles

Safety and Efficacy of Transplantation with Allogeneic Skin Tumors to Treat Chemically-Induced Skin Tumors in Mice

BackgroundTransplantation with allogeneic cells has become a promising modality for cancer therapy, which can induce graft-versus-tumor (GVT) effect. This study was aimed at assessing the safety, efficacy, and tissue type GVT (tGVT) response of transplantation with allogeneic skin tumors to treat chemically-induced skin tumors in mice.Material/MethodsFVB/N and ICR mice were exposed topically to chemicals to induce skin tumors. Healthy ICR mice were transplanted with allogeneic skin tumors from FVB/N mice to test the safety. The tumor-bearing ICR mice were transplanted with, or without, allogeneic skin tumors to test the efficacy. The body weights (BW), body condition scores (BCS), tumor volumes in situ, metastasis tumors, overall survival, and serum cytokines were measured longitudinally.ResultsTransplantation with no more than 0.03 g allogeneic skin tumors from FVB/N mice to healthy ICR mice was safe. After transplantation with allogeneic skin tumors to treat tumor-bearing mice, it inhibited the growth of tumors slightly at early stage, accompanied by fewer metastatic tumors at 24 days after transplantation (21.05% vs. 47.37%), while there were no statistically significant differences in the values of BW, BCS, tumor volumes in situ, metastasis tumors, and overall survival between the transplanted and non-transplanted groups. The levels of serum interleukin (IL)-2 were significantly reduced in the controls (P<0.05), but not in the recipients, which may be associated with the tGVT response.ConclusionsOur results suggest that transplantation with allogeneic skin tumors is a safe treatment in mice, which can induce short-term tGVT response mediated by IL-2.

Tolerogenic Phenotype of IFN-γ–Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO–Kynurenine/AhR–IDO Loop

Previous studies demonstrated that IL-12-driven antitumor activity is short-circuited by a rapid switch in dendritic cell (DC) function from immunogenic to tolerogenic activity. This process was dependent on IFN-γ and the tolerogenic phenotype was conferred by IDO. Extended monitoring of IDO(+) DC in the tumor-draining lymph nodes of IL-12 plus GM-CSF-treated tumor-bearing mice revealed that whereas IFN-γ induction was transient, IDO expression in DC was maintained long-term. An in vitro system modeling the IFN-γ-mediated change in DC function was developed to dissect the molecular basis of persistent IDO expression in post-IL-12 DC. Stimulation of DC with IFN-γ and CD40L resulted in rapid induction of IDO1 and IDO2 transcription and recapitulated the in vivo switch from immunogenic to tolerogenic activity. Long-term maintenance of IDO expression was found to be independent of exogenous and autocrine IFN-γ, or the secondary cytokines TGF-β, TNF-α, and IL-6. In contrast, both IDO enzymatic activity and IFN-γ-induced AhR expression were required for continued IDO transcription in vitro and in vivo. Addition of the tryptophan catabolite kynurenine to DC cultures in which IDO activity was blocked restored long-term IDO expression in wild-type DC but not in AhR-deficient DC, establishing the central role of the kynurenine-AhR pathway in maintaining IDO expression in tolerogenic DC. These findings shed further light on the cellular and molecular biology of the post-IL-12 regulatory rebound and provide insight into how feedback inhibitory mechanisms dominate in the long-term.

Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin.

In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo, we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo. These anticancer effects correlated with a reduced number of CSF-1Rpos cells, in tumors excised from the treated mice. Depletion of the CD45pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components.

Abstract 4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells

Abstract Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. Using a HPV-expressing murine tumor model (C3), we found that treatment with mCPA (20 mg/kg/day PO) in combination with a DPX peptide vaccine caused selective enrichment of antigen-specific CD8+ T cells, resulting in increased immune responses detected by IFN-ã ELISPOT and in vivo cytotoxicity assay. The combination provided long term-control of tumors when initiated within one week of tumor implantation, however efficacy was limited in mice bearing advanced tumors. Antigen-specific CD8+ T cells could be detected infiltrating advanced tumors by flow cytometry, along with increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and PD-1 blockade (with anti-PD-1 or anti-PD-L1) resulted in tumor control of established tumors which were not successfully treated with antibody monotherapy. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRâ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRâ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRâ sequences (p&amp;lt;0.05). We conclude that anti-PD-1 therapy can enhance the efficacy of vaccine immunotherapy by promoting the activity and expansion of antigen-specific T cells within the TME. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac. Assessment of T cell clonality within the tumor may be an important biomarker for immunomodulatory treatments. Citation Format: Genevieve Weir, Olga Hrytsenko, Tara Quinton, Mohan Karkada, Neil L. Berinstein, Marianne Stanford, Marc Mansour. Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells. [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 4903.

Abstract 1485: Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment

Abstract CD40-activating immunotherapy has potent anti-tumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by accumulation of immunosuppressive cells in the tumor and by endothelial anergy induced by pro-angiogenic growth factors. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib significantly decrease tumor growth and improve survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb lead to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, as evidenced by enhanced CD86 expression, while sunitinib treatment reduced vessel density. CD11b+Gr1+ myeloid derived suppressor cells accumulated in the tumor draining lymph nodes after anti-CD40 mAb treatment. This effect was reversed by co-treatment with sunitinib. Tumor endothelial expression of ICAM1 and VCAM1 adhesion molecules were increased only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced tumoral infiltration of CD8+ T-lymphocytes. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exert potent complementary antitumor effects mediated by dendritic cell activation, reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T cells. Citation Format: Luuk van Hooren, Maria Georganaki, Hua Huang, Sara Mangsbo, Anna Dimberg. Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment. [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 1485.

Abstract 1080: Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice

Abstract Introduction Small-interfering RNA (siRNA) is an attractive option for delivering precision therapy to cancer patients because of the potential for highly-specific control of gene expression. Attempts to utilize siRNA in vivo have been hampered by its short circulating half-life, limited cellular uptake, and cellular confinement within endosomes. To overcome these barriers, we packaged siRNA within serum-stable, cell-penetrating, and endosomolytic peptide-based nanoparticles (NPs) to deliver siRNA to human and mouse pancreas and colorectal cancers. In a small pilot study, twice weekly administration of KRAS siRNA-NP to mice bearing human colorectal cancers significantly reduced tumor growth compared to controls. Methods NP uptake was assessed in cell lines utilizing fluorescent siRNA-NPs in combination with fluorescence microscopy and flow cytometry. Cell lines were incubated with KRAS-siRNA NP and KRAS knockdown was assessed by quantitative PCR. Mice bearing tumors derived from these cell lines were injected intravenously with fluorescent NP, and localization and uptake were assessed with an in vivo imaging system (IVIS) and flow cytometry. Immune deficient mice were inoculated subcutaneously with a human colorectal cancer and half were treated for 4 weeks with twice weekly IV injections of KRAS siRNA-NP (1 nmol KRAS siRNA per dose). Results On average, fluorescent siRNA was detected in more than 93% of human and mouse pancreas and colorectal cancer cells (n = 5 cell lines) after 24 hours of incubation. Exposure of colorectal cancer cells to KRAS siRNA-NP in vitro produced a conservatively-estimated 70% downregulation of KRAS. Tumors derived from all cell lines were strongly fluorescent 2 hours after IV injection of fluorescent NP and signal persisted beyond 30 hours. The liver and kidneys also expressed fluorescent signal by IVIS. 86% of tumor cells derived from tumors exposed to fluorescent-NP by IV injection expressed fluorescent signal 24 hours post-injection. Only 18% and 14% of liver and kidney cells, respectively, were fluorescent. Compared to control mice, tumor size was reduced in mice bearing colorectal cancer that received KRAS-siRNA NP IV injections. Eighteen days after initiation of treatment, tumor volumes in the control and treatment groups were 229.5 mm3 and 460.25 mm3 respectively (p &amp;lt; 0.05). Hematologic, liver and kidney function tests revealed no differences between the treatment and control groups. Conclusions Peptide NPs are rapidly and efficiently taken up by human and mouse pancreas and colorectal cancer cells both in vitro and in vivo. Incubation of cancer cells with peptide NP-packaged KRAS-siRNA reduces KRAS expression in vitro. Treatment of tumor-bearing mice with KRAS-siRNA NP leads to a statistically significant reduction in tumor growth compared to control mice. This delivery platform overcomes traditional limitations of siRNA and may be generalizable to target other putative drivers of tumor progression. Citation Format: Matthew Strand, Hua Pan, Xiuli Zhang, Julie Grossman, Peter Goedegebuure, Timothy Fleming, William E. Gillanders, Samuel A. Wickline, Ryan C. Fields. Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice. [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 1080.

Abstract 1245: RRx-001 is effective in temozolomide-sensitive and resistant GBM

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive malignancies in humans, being highly invasive and drug-resistant, limiting the effectiveness of surgery, chemotherapy and radiotherapy. Concurrent Temozolomide (TMZ) and radiation is the standard of care for newly diagnosed GBM patients, however, patients respond poorly and prognosis remains dismal. TMZ response is limited to patients with hypermethylation of the methylguanine methyltransferase (MGMT) promoter. Despite recent advances in therapeutic strategies, treating GBM is still an unsolved clinical challenge requiring the investigation of novel approaches with better responses over approved therapies. RRx-001 is a novel ROS-mediated systemically non-toxic chemo-sensitizing epigenetic agent with vascular normalizing properties under investigation in Phase II clinical trials in brain metastases and GBM. We assessed whether RRx-001 is an effective approach for treating GBM by testing its effects alone and in combination with TMZ both in vitro as well as in GBM mouse models. RRx-001 induced cytotoxicity in human TMZ-sensitive as well as resistant patient-derived GBM cell lines. In addition, its effect in TMZ-sensitive GBM cells was greater than the one induced by TMZ alone. We then asked whether the in vitro effects of RRx-001 translated to therapeutic efficacy in vivo. To this end, we injected mice intracranially with a TMZ-resistant murine mesenchymal GBM cells, and treated tumor-bearing mice with RRx-001 alone and in combination with TMZ. RRx-001 treatment alone modestly prolonged survival compared to control cohorts, whereas the combination of RRx-001 and TMZ substantially prolonged survival compared to controls and either treatment alone. Moreover, histological evaluation of GBM tumors revealed that combination of RRx-001 and TMZ induced vessel hyperdilation and apoptosis compared to RRx-001 alone. Thus, in vivo RRx-001 combined to TMZ may also indirectly regulate tumor growth by modifying the tumor stroma. These data suggest that TMZ may still affect the microenvironment in TMZ-resistant tumors and that RRx-001 enhances these effects by both directly and indirectly restricting GBM growth, making it a promising therapeutic agent alone and in combination with TMZ. Citation Format: Veronica Steri, Bryan Oronsky, Jan Scicinski, Gabriele Bergers. RRx-001 is effective in temozolomide-sensitive and resistant GBM. [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 1245.

Abstract 3770: HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075

Abstract Bromodomain and extra-terminal (BET) family are dual bromodomain-containing proteins that play an important role in transcription regulation. ABBV-075 is a small molecule BET bromodomain inhibitor currently in phase 1 clinical trials. Here we report the identification of Hexim1 and other BET-responsive genes as robust pharmacodynamic markers for monitoring ABBV-075 target engagement in tumors and in surrogate tissues such as whole blood and skin. Transcription profiling of cancer cell lines for their responses to BET inhibitors identified a set of 9 BET-regulated genes, including Hexim1, that responded to BET inhibition across cancer cell lines and in xenograft tumors. Further characterization of BET-responsive genes in surrogate tissues such as mouse skin, mouse/human PBMCs and whole blood revealed that the mRNA level of Hexim1 exhibited the best response to BETi treatment across different settings. A dose-dependent increase of Hexim1 expression was detected in the whole blood of ABBV-075 treated tumor bearing mice, and the Hexim1 response was closely correlated with the plasma drug concentration and largely reflected the anti-tumor efficacy at various dose levels of ABBV-075. Hexim1 is part of a protein/RNA complex that sequesters pTEFb and prevents its recruitment by BRD4. It also reportedly mediates the anti-proliferative activity of BRD4 in AML. Taken together, Hexim1 could serve as a functional relevant pharmacodynamic marker for monitoring ABBV-075 target engagement in animal models and in the clinical setting. Citation Format: Xiaoli Huang, Xiaoyu Lin, Leiming Li, Rongqi Wang, Lisa Roberts, Paul Hessler, Tamar Uziel, Lloyd Lam, Terry Magoc, Daniel H. Albert, Steven W. Elmore, Guowei Fang, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075. [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 3770.

Abstract 1496: Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes

Abstract Objectives: Given the heterogeneity of ovarian cancer, it is imperative to identify subtype-directed treatments. The novel antibody NEO-201 targets a specific tumor-associated antigen (TAA) expressed on some ovarian and uterine malignancies, providing a tumor-directed approach. Here, we aimed to identify the cancer subtypes which express the NEO-201 target and demonstrate its cytotoxic effects in vitro and in mouse models of ovarian cancer. Methods: NEO-201 is a genetically humanized novel monoclonal antibody developed through vaccines with TAAs. This antibody targets malignant tissues that express tumor-specific epitopes in membrane-anchored protein CEACAM-6. We performed immunohistochemistry (IHC) on tissue microarrays from formalin-fixed paraffin-embedded subtyped ovarian and uterine cancers to estimate the incidence of cancers expressing the NEO-201 target. Ovarian and uterine cell line pellet arrays were stained by IHC to identify in vitro models. We examined the cytotoxicity of NEO-201 in two high- and three low-expressing cell lines using Calcein AM cell viability assays alone and with purified natural killer (NK) cells with and without IL-2 stimulation. Studies of NEO-201 in ovarian cancer mouse models are ongoing. Results: IHC of NEO-201 in tissue microarrays demonstrated 51% and 12% reactivity in uterine and ovarian samples, respectively. Similar expression patterns were identified in representative cell lines by IHC and Western blot. NEO-201 killed cell lines expressing its target in the range of 0.5-20μg/mL. Over 3 weeks, NEO-201 treatment of tumor-bearing mice demonstrated control of tumor growth with 3 doses of 250μg NEO-201 and tumor regression with 100μg in combination with IL2-stimulated PBMC. Studies are ongoing to further investigate NEO-201 cytotoxicity in ovarian cancer mouse models as well as identify the mechanism of tumor cell death and the target epitope of NEO-201. Conclusions: NEO-201, a novel monoclonal antibody, specifically targets epithelial malignancies including ovarian and uterine cancer. This potentially therapeutic antibody demonstrates tumor-specific cytotoxicity in cancer cell lines expressing its target in vitro and in xenografts with IL2-stimulated PBMC, suggesting both antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity mechanisms are involved in tumor inhibition and regression. These studies lay the groundwork for future examination of TAA-directed therapy for ovarian or uterine malignancies. Citation Format: Monica K. Neuman, Lidia Hernandez, Xue­-Ping Wang, Olga Saric, Alex Dubeykovskiy, Philip Arlen, Christina M. Annunziata. Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes. [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 1496.

Abstract 2439: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150 increases the chemosensitivity and decreases tumor-initiating potential of neuroblastoma cells

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The long-term, event-free survival of high-risk NB remains ∼ 50% despite intensive multi-modality treatments. Activated JAK/STAT3 pathway plays an important role in many human cancers. In NB patients, cytokines activating STAT3 have been associated with poor patient outcome and have been implicated in the survival of a rare population of NB tumor initiating cells. Thus targeting STAT3 may be a promising therapeutic strategy for high-risk NB. To evaluate the biologic consequences of genetic targeting of STAT3, we assessed the effects of inhibition of STAT3 in NB cell lines containing a tetracycline (Tet)-inducible STAT3 expression plasmid. Additionally we evaluated pharmacogenomic inhibition of STAT3 using AZD9150, a generation 2.5, 16-nucleotide, antisense oligonucleotide (ASO) that is now in Phase I/II clinical trials. Studies were conducted in 3 representative NB cell line models (AS (MYCN-WT) and NGP and IMR32 (MYCN-Amplified)). Both the Tet-inducible STAT3 shRNA and AZD9150 reduced endogenous STAT3 mRNA and protein levels causing decreased transcription and expression of STAT3 target genes, such as CyclinD1, D3, and MYC/MYCN. In functional in vitro studies Tet-inducible STAT3 shRNA and AZD9150 decreased NB cell migration and clonogenicity in soft agar. In vivo, STAT3 inhibition by Tet-inducible STAT3 shRNA or AZD9150 alone had little effect on the growth of established tumors nor did it alter the survival of tumor-bearing mice, despite decreases in STAT3, P-STAT3 and target gene expression in xenografts from AZD9150-treated mice compared to those from ASO-treated mice. To assess whether inhibition of STAT3 altered the tumor initiating potential of NB cells, NB tumor xenograft cells from ASO or AZD9150 treated mice were re-implanted and secondary tumor growth assessed. At 200,000 and 20,000 ASO-treated NB cells, 100% of mice had tumors while only 40 and 20%, respectively of AZD9150-treated mice had tumors. These results indicate that the inhibition of STAT3 decreased the frequency of tumor initiating cells in the NB xenograft from AZD9150 treated mice. Since tumor initiating or stem-like cells are frequently more resistant to cytotoxic agents, we next evaluated a combination therapy with cisplatin. We found that in vitro either genetic shSTAT3 or AZD9150 mediated pharmacogenomic inhibition of STAT3 significantly increased the sensitivity of NB cells to cisplatin. Furthermore, in established NB tumors xenografts, the combination of STAT3 inhibition with cisplatin caused a 30% decrease in tumor size (P = 0.0092) and increased the survival of AZD9150 treated tumor-bearing mice compared to ASO-treated mice (P = 0.026). Our study supports the development of strategies targeting STAT3 in combination with conventional chemotherapy for patients with high-risk NB. Citation Format: Seiichi Odate, Shuang Yan, Veronica Veschi, Norris Lam, Zhihui Liu, Carol J. Thiele. Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150 increases the chemosensitivity and decreases tumor-initiating potential of neuroblastoma cells. [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 2439.

Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment.

CD40-activating immunotherapy has potent antitumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by immunosuppressive cells in the tumor and by endothelial anergy inhibiting recruitment of T-cells. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib decreased tumor growth and improved survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb led to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, while sunitinib treatment reduced vessel density and decreased accumulation of CD11b+Gr1+ myeloid derived suppressor cells. The expression of ICAM-1 and VCAM-1 adhesion molecules was up-regulated on tumor endothelial cells only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced intratumoral infiltration of CD8+ cytotoxic T-cells. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exerts potent complementary antitumor effects mediated by dendritic cell activation, a reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T-cells.

PEG-rIL-10 treatment decreases FoxP3+ Tregs despite upregulation of intratumoral IDO

ABSTRACTIL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4+ T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO+FoxP3+CD4+ Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFNγ and CD8+ T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFNγ. We have continued to assess PEG-rIL-10's pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFNγ-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3+CD4+ T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGFβ/IL-2-dependent in vitro polarization of FoxP3+CD4+ Tregs and potentiates IFNγ+T-bet+CD4+ T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3+CD4+ Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10's potent antitumor immunity.

Bladder tumor-targeted delivery of pro-apoptotic peptide for cancer therapy

The overall prognosis of conventional chemotherapy for the treatment of bladder cancer is poor and reduction of its systemic side effects remains an unsolved issue. Targeted therapy for bladder cancer could improve therapeutic efficacy and reduce side effects. This study investigated a hybrid peptide (named Bld-1-KLA) composed of the CSNRDARRC peptide (Bld-1), which binds to bladder tumor cells, and the d-KLAKLAKKLAKLAK (KLA) peptide, which disrupts mitochondrial membrane and induces apoptotic cell death, as a bladder cancer-targeted therapeutic agent. Bld-1-KLA selectively bound to HT1376 bladder tumor cells and efficiently internalized into the cells but not to other types of tumor and normal cell lines. Bld-1-KLA exerted cytotoxic effects selectively to HT1376 cells (LC50=41.5μM), but not to other types of cells. Pretreatment of cells with Bld-1 inhibited the binding and cytotoxicity by Bld-1-KLA in HT1376 cells. It induced apoptosis of bladder tumor cells, while Bld-1 or KLA alone showed much lesser effect on apoptosis, and was co-localized in mitochondria. Bld-1-KLA was stable up to 24h in serum. In vivo fluorescence imaging showed that homing of Bld-1-KLA in the tumor in HT1376 tumor-bearing nude mice was greater than that of the control peptide-KLA after intravenous injection. Treatment of tumor-bearing mice with Bld-1-KLA, compared to the control peptide-KLA, induced apoptosis of tumor cells and inhibited tumor growth more efficiently. No significant side effects on body weight and the liver and myeloid function were observed in mice treated with Bld-1-KLA. These results suggest that Bld-1-KLA is a promising therapeutic agent for targeted therapy of bladder cancer.

PO-33 - Heparin suppresses progression of small cell lung cancer (SCLC) in an orthotopic mouse model

Lung cancer is the leading cause of cancer-related death worldwide. Small cell lung cancer (SCLC) is thereby a highly aggressive neuroendocrine carcinoma representing about 15% of all lung cancer cases. Due to the highly metastatic behavior and multidrug resistance, the long-term survival of patients is very low. Current clinical studies revealed an increased survival of SCLC patients treated with heparin. Thus, the role of heparin in SCLC progression was analyzed with the focus on cell adhesion, cell survival and metastasis formation. Heparins were tested for their capacities to alter migration, adhesion and viability of SCLC cells in vitro as well as tumor growth and metastasis formation in vivo. Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) both strongly inhibited migration as well as adhesion of SCLC cells to fibronectin and stromal cells. In addition, Heparin induced cellular apoptosis and also increased apoptotic effects of conventional chemotherapeutics in vitro. To investigate the role of LMWH on metastasis formation in vivo, an orthotopic xenograft mouse model with spontaneous metastasis formation has been established. The primary tumors in this mouse model show a marked capacity to metastasize to characteristic distant organs, reflecting advanced steps of malignant progression. Treatment of tumor-bearing mice with LMWH suppressed progression of SCLC. Administration of LMWH in addition to the conventional treatment might reduce metastasis formation and development of chemoresistance, leading to an improved survival rate of patients suffering from SCLC.

M011L-deficient oncolytic myxoma virus induces apoptosis in brain tumor-initiating cells and enhances survival in a novel immunocompetent mouse model of glioblastoma

Myxoma virus (MYXV) is a promising oncolytic agent and is highly effective against immortalized glioma cells but less effective against brain tumor initiating cells (BTICs), which are believed to mediate glioma development/recurrence. MYXV encodes various proteins to attenuate host cell apoptosis, including an antiapoptotic Bcl-2 homologue known as M011L. Such proteins may limit the ability of MYXV to kill BTICs, which have heightened resistance to apoptosis. We hypothesized that infecting BTICs with an M011L-deficient MYXV construct would overcome BTIC resistance to MYXV. We used patient-derived BTICs to evaluate the efficacy of M011L knockout virus (vMyx-M011L-KO) versus wild-type MYXV (vMyx-WT) and characterized the mechanism of virus-induced cell death in vitro. To extend our findings in a novel immunocompetent animal model, we derived, cultured, and characterized a C57Bl/6J murine BTIC (mBTIC0309) from a spontaneous murine glioma and evaluated vMyx-M011L-KO efficacy with and without temozolomide (TMZ) in mBTIC0309-bearing mice. We demonstrated that vMyx-M011L-KO induces apoptosis in BTICs, dramatically increasing sensitivity to the virus. vMyx-WT failed to induce apoptosis as M011L protein prevented Bax activation and cytochrome c release. In vivo, intracranial implantation of mBTIC0309 generated tumors that closely recapitulated the pathological and molecular profile of human gliomas. Treatment of tumor-bearing mice with vMyx-M011L-KO significantly prolonged survival in immunocompetent-but not immunodeficient-mouse models, an effect that is significantly enhanced in combination with TMZ. Our data suggest that vMyx-M011L-KO is an effective, well-tolerated, proapoptotic oncolytic virus and a strong candidate for clinical translation.

Abstract B23: Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors

Abstract Kinases are an important class of targets for a number of therapeutic indications, and currently, kinase inhibitors constitute a significant fraction of recently approved drugs, particularly for cancer. Using a probe-based chemoproteomics platform, we have demonstrated the utility of the platform to monitor the interactions between inhibitors and native kinases. Herein, we profile kinase inhibitors in cell lines either sensitive or resistant to the compound for various periods of time. We observe that at early time points, inhibitor profiles for a compound are qualitatively similar in both the sensitive and resistant cells, suggesting the compound is capable of engaging its direct targets in both cell types. In contrast, at later time points, additional kinases are observed affected by compound treatment. We infer that these kinases do not directly bind the compound but rather reflect secondary effects that result from the engagement of the primary targets (pathway effects). Significantly, the pathway effects are only observed in the sensitive cells and not the resistant cells. Kinases affected through these pathway effects include kinases known to be critical for cell cycle progression including CDK1, Aurora A and B, PLK1, and MASTL. Finally, we demonstrate that during the treatment of tumor-bearing mice with an inhibitor, in addition to inhibition of the direct targets, pathway effects are observed recapitulating the results from the cell-based studies. Citation Format: Tyzoon K. Nomanbhoy, Heidi E. Brown, Jiangyue Wu, Subha Vogeti, Arwin Aban, Wendy Grant, Alemayehu Senait, Shuzhen Wu, Christa Dias, Geeta Sharma. Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B23.

Abstract B66: Blockade of PD-1 signaling in tumor-associated dendritic cells results in compensatory IL-10 release maintaining immune suppression in ovarian cancer microenvironments.

Abstract PD-1 and PD-L1 form a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells, PD-1 has also been shown to be expressed on and hence impact the functions of cells of innate arm, such as dendritic cells (DCs), primarily at the tumor site but not in the periphery. Our aim in this study was to identify tumor-associated factor(s) responsible for PD-1 regulation on DCs and investigate how DCs in the tumor microenvironment respond to anti-PD-1-based immunotherapy. Both bone marrow-derived dendritic cells (BMDCs) as well as murine ovarian tumor ascites-derived DCs were used in the studies. Using in vitro cultures, we show that the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer, is potent regulator of PD-1 expression on DCs. Based on inhibition and siRNA knockdown studies; we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Treatment of DCs with IL-10 also led to an increase in expression of PD-L1 on surface of DCs as well as an increase in release of soluble PD-L1. Antibody-mediated blockade of PD-1 on DCs led to greatly increased production of IL-10 (&amp;gt; 4 fold) in cultures in vitro. Furthermore, treatment of tumor bearing mice with anti-PD-1 antibody led to a significant increase of IL-10 in ascites (&amp;gt; 6 fold) and periphery (&amp;gt; 4 fold). This compensatory release of IL-10 correlated with a further increase in PD-1 expression on DCs both in vitro and in vivo. In in vivo studies, while the blockade of PD-1 or IL-10 neutralization as monotherapy were ineffective, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ovarian tumor bearing mice; leading to a decrease in tumor growth and a significant increase in survival. These results show that the IL-10 and PD-1 pathways intersect with DCs in the tumor microenvironment. PD-1 blockade on DCs leads to compensatory release of IL-10 resulting in the maintenance of immunosuppression, and a combination regimen of PD-1 blockade and IL-10 neutralization has therapeutic benefit in ovarian tumors. These results serve as catalyst to explore the feasibility/efficacy of exploiting these targets in clinical settings for treatment of human ovarian cancer. Citation Format: Purushottam Lamichhane, Lavakumar Karyampudi, Barath Shreeder, James Krempski, Deborah Bahr, Matthew Block, Keith Knutson. Blockade of PD-1 signaling in tumor-associated dendritic cells results in compensatory IL-10 release maintaining immune suppression in ovarian cancer microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B66.

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

Complex of C60 Fullerene with Doxorubicin as a Promising Agent in Antitumor Therapy

The main aim of this work was to evaluate the effect of doxorubicin in complex with C60 fullerene (C60 + Dox) on the growth and metastasis of Lewis lung carcinoma in mice and to perform a primary screening of the potential mechanisms of C60 + Dox complex action. We found that volume of tumor from mice treated with the C60 + Dox complex was 1.4 times less than that in control untreated animals. The number of metastatic foci in lungs of animals treated with C60 + Dox complex was two times less than that in control untreated animals. Western blot analysis of tumor lysates revealed a significant decrease in the level of heat-shock protein 70 in animals treated with C60 + Dox complex. Moreover, the treatment of tumor-bearing mice was accompanied by the increase of cytotoxic activity of immune cells. Thus, the potential mechanisms of antitumor effect of C60 + Dox complex include both its direct action on tumor cells by inducing cell death and increasing of stress sensitivity and an immunomodulating effect. The obtained results provide a scientific basis for further application of C60 + Dox nanocomplexes as treatment agents in cancer chemotherapy.

Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.