Tumor-free Mice Research Articles

Synergistic tumoricidal effect of combined hPD-L1 vaccine and HER2 gene vaccine

Immunotherapy is gathering momentum as a kind of important therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only in a small subset of patients. Combination therapies targeting multiple pathways often can augment an immune response to improve survival further. Here, the tumoricidal effects of the dual hPD-L1(human programmed cell death ligand 1) vaccination/HER2(human epidermal growth factor receptor 2) gene vaccination immunotherapy against the established HER2-expressed cancers were observed. Animals treated with combination therapy using hPD-L1 vaccine and HER2 gene vaccine had significantly improved survival in a mammary carcinoma model. We observed an increase in tumor growth inhibition following treatment. The percentage of the tumor-free mice (%) was much higher in the combined PD-L1/HER2 group. Furthermore, under the tumor-burden condition, hPD-L1 vaccine enhanced humoral immunity of HER2 gene vaccine. And the combination treatment increased the IFN-γ-producing effector T cells. Additionally, splenocytes from the combined PD-L1/HER2 group immunized mice possessed higher CTL activity. Notably, vaccination with combination therapy induced a significant decrease in the percentage of CD4+CD25+ Treg cells. Collectively, these data demonstrate that PD-L1/HER2 gene vaccine combination therapy synergistically generates marked tumoricidal effects against established HER2-expressing cancers.

Fraction From Lycium barbarum Polysaccharides Reduces Immunotoxicity and Enhances Antitumor Activity of Doxorubicin in Mice.

The aim of the present study was to investigate whether fraction from Lycium barbarum polysaccharide (LBP) could reduce immunotoxicity and enhance antitumor activity of doxorubicin (Dox) in mice. A water-soluble LBP fraction, designated LBP3, was isolated from edible Chinese herbal Lycium barbarum and used in this study. To investigate the effect of LBP3 on Dox-induced immunotoxicity, tumor-free mice were used and treated with either normal saline, Dox, or Dox plus LBP3. To investigate the effect of LBP3 on antitumor activity of Dox, H22 tumor-bearing mice were used and treated with either normal saline, Dox, LBP3, or Dox plus LBP3. The results showed that LBP3 did not protect against the body weight loss caused by Dox, but it promoted the recovery of body weight starting at day 5 after Dox treatment in tumor-free mice. LBP3 also improved peripheral blood lymphocyte counts, promoted cell cycle recovery in bone marrow cells, and restored the cytotoxicity of natural killer cells. Furthermore, in H22 tumor-bearing mice, LBP3 enhanced antitumor activity of Dox and improved peripheral blood lymphocyte counts and the cytotoxicity of splenocytes. In brief, our results demonstrated that LBP3 could reduce the immunotoxicity and enhance antitumor activity of Dox.

Combining ultrasound and intratumoral administration of doxorubicin-loaded microspheres to enhance tumor cell killing

Melanoma is an incurable disease for which alternative treatments to chemotherapy alone are sought. Here, using a melanoma model, we investigated the antitumor potential of combining ultrasound (US) with poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with doxorubicin (DOX). The aim was to achieve synergistic tumoricidal activity through direct and indirect US-mediated damage of tumor cells combined with sustained and potentially controllable release (when combined with US) of DOX from microspheres. An in vitro release assay demonstrated an ability of US to affect the release kinetics of DOX from DOX-loaded PLGA microspheres by inducing a 12% increase in the rate of release. In vitro viability assays demonstrated that combining US with DOX-loaded PLGA microspheres resulted in synergistic tumor cell (B16-F10 melanoma cells) killing. Melanoma-bearing mice were treated intratumorally with DOX (8 µg)-loaded microspheres and subjected to US treatment at the tumor site. This treatment could significantly extend survival (mean survival (MS) = 22.1 days) compared to untreated mice (MS = 10.4 days) and most other treatments, such as blank microspheres plus US (MS = 11.5 days) and DOX (8 µg)-loaded microspheres alone (MS = 13 days). The findings that immune checkpoint blockade did not significantly extend survival of mice treated with DOX (8 µg)-loaded microspheres plus US, and that tumor-free (“cured”) mice were not protected from subsequent tumor rechallenge suggests minimal involvement of the adaptive immune response in the observed antitumor activity. Nevertheless, the synergistic increase in survival of melanoma-challenged mice treated with the combination of US and DOX-loaded microspheres implicates such a treatment methodology as a promising additional tool for combatting otherwise currently incurable cancers.

Cyclophosphamide eradicates murine immunogenic tumor coding for a non-self-antigen and induces antitumor immunity.

Although the majority of cancers respond to chemotherapy, most cancer types relapse, at least in part, due to the poor immunogenicity of most tumor. We have reported before that treatment of tumor bearing mice with a combination of the anti-cancer chemotherapy cyclophosphamide (CTX) and immunotherapy can result in complete tumor regression using T-cell receptor (TCR) transgenic CD8+ T cells specific to antigens. This study aimed to determine whether chemotherapy can cure immunogenic tumor which expresses non-self-tumor antigen and result in antitumor immunity. Either EL4 cell line, a poorly immunogenic thymoma, or EG7, a clone of EL4 cells transfected with ovalbumin (OVA), as a non-self-antigen were inoculated subcutaneously into wild type or splenectomized C57BL/6 mice and then treated once with intraperitoneal (i.p.) injection of 4 mg CTX/mouse. In certain experiments, the mice were rechallenged with the same tumor type 1–2 months after the primary challenge. Treatment of EL4 bearing mice with CTX induced transient antitumor effect followed by tumor progression. Interestingly, however, treatment of EG7-bearing mice with CTX resulted in regression of early and advanced tumors. EG7 tumor-free mice rejected the second and the third challenges with EG7 cells, but not with challenge EL4 cells. These antitumor effects did not require spleen, since splenectomized mice showed similar antitumor effects of CTX on EG7 cells. Taken together, these data indicate that expression of non-self-antigen by poorly immunogenic tumor might be a reliable means to increase its immunogenicity and its response to chemotherapy.

Abstract A62: In vivo electroporation improves the immune responses induced by a DNA vaccine and confers complete antitumor protection against HPV-associated tumors

Abstract Cervical cancer associated with persistent papilloma virus infections (HPV) is a public health problem and represents the third most frequent type of cancer in women. Our group developed a DNA vaccine against HPV-induced tumors based on the expression of the HPV-16 E7 oncoprotein genetically fused to the HSV-1 glycoprotein D (gD) (pgDE7h). This vaccine is capable of promoting antitumoral effects against tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells) and induces specific cellular immune responses. In view of the low success rate of DNA vaccines in clinical trials, in vivo electroporation (EP) is a promising strategy for plasmid delivery, leading to increased cellular transfection and enhanced immune responses. In this study, C57BL/6 mice were inoculated with TC-1 cells subcutaneously and immunized 3 days later with pgDE7h intramuscularly, associated or not with EP. Immune responses were analyzed 14 days after vaccination and tumor growth was monitored for 60 days. Similar antitumor effects were obtained with 10-fold less DNA associated with EP when compared to the group that was inoculated only with DNA, reaching around 20% of tumor-free mice. Dose-response curves indicated that maximum protective immunity was achieved with 50 ug of vaccine inoculated with EP, promoting 100% of antitumor protection. This group also had enhanced frequencies of cytotoxic lymphocytes expressing the degranulation marker CD107a and high in vivo cytotoxic activities. Besides that, a delayed vaccination performed 10 days after the TC-1 injection led to 70% of tumor-free mice until the end of the observed period. Collectively, these data confirmed that EP can be used as an efficient method of DNA vaccine delivery and should be further studied to improve the chances of cancer vaccine success in clinical trials. Citation Format: Natiely Silva Sales, Mariana de Oliveira Diniz, Jamile Ramos Silva, Mariângela de Oliveira Silva, Bruna F. M. M. Porchia, Luana R. M. M. Aps, Luis Carlos Souza Ferreira. In vivo electroporation improves the immune responses induced by a DNA vaccine and confers complete antitumor protection against HPV-associated tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A62.

Dexamethasone exacerbates cytotoxic chemotherapy induced lethargy and weight loss in female tumor free mice

ABSTRACTCytotoxic chemotherapy can induce a systemic inflammatory response which has been proposed to be an underlying mechanism of cancer treatment related fatigue. Dexamethasone, a synthetic glucocorticoid that has potent anti-inflammatory effects, is incorporated into chemotherapy regimens to prevent chemotherapy-induced nausea and vomiting (CINV). The purpose of this study was to determine whether by suppressing cytotoxic chemotherapy-induced inflammation, dexamethasone could ameliorate chemotherapy induced fatigue/lethargy in tumor free mice. The effect of dexamethasone (DEX) on Cytoxan-Adriamycin (CA)-induced inflammation was assessed by measuring circulating levels of IL-1β, TNF-α, IL-6, GCSF, KC, and MCP-1 twenty-four-hours post CA injection. Decline in voluntary wheel running activity (VWRA) from baseline (used as a proxy for fatigue/lethargy), body weight and composition, and food intake were monitored in mice administered four cycles of CA every two weeks with or without DEX. CA increased circulating levels of IL-6, GCSF, KC, and MCP-1 and caused a rapid decline in VWRA and body weight immediately following CA-injection. Although the acute CA-induced decline in VWRA and body weight was not evident in mice administered CA + DEX, DEX alone had a suppressive effect on VWRA, and body weight continued to decline in mice administered both CA and DEX while it returned to baseline in CA-treated mice. CA or DEX alone had no long term impact on VWRA but DEX exacerbated lethargy and weight loss in CA-treated mice. Despite dampening the systemic inflammatory response to chemotherapy, dexamethasone failed to ameliorate acute or long term chemotherapy related fatigue/lethargy. Our pre-clinical findings suggest that supportive therapies like dexamethasone used to acutely control nausea and vomiting in cancer patients may actually contribute to overall symptom burden in cancer patients.

Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas

ObjectiveThe response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those...

Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors

Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan cured mice bearing small (∅ ≈ 0.5 cm), but not large (∅ ≈ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed ‘TheraVac’ consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.

Nano-pulse stimulation induces potent immune responses, eradicating local breast cancer while reducing distant metastases.

Nano-pulse stimulation (NPS) as a developing technology has been studied for minimally invasive, nonthermal local cancer elimination for more than a decade. Here we show that a single NPS treatment results in complete regression of the poorly immunogenic, metastatic 4T1-Luc mouse mammary carcinoma. Impressively, spontaneous distant organ metastases were largely prevented, even in those animals with incomplete tumor regression. All tumor-free mice were protected from secondary tumor cell challenge, demonstrating a vaccine-like effect. NPS treatment induced antitumor immunity, long-term memory T cells, destruction of tumor microenvironment and reversal of the massive increase of immune suppressor cells in the tumor microenvironment and blood. NPS-treated 4T1 cells exhibited release of damage-associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, and activated dendritic cells. Those findings suggest that NPS is a potent immunogenic cell death inducer that elicits antitumor immunity to prevent distant metastases in addition to local tumor eradication.

Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Opioids modulate the tumor microenvironment with potential functional consequences for tumor growth and metastasis. We evaluated the effects of morphine administration on the circulating proteolytic profile of tumor-free mice. Serum from morphine-treated (1 or 10 mg/kg, i.p. every 12 h) or saline-treated mice was collected at different time points and tested ex vivo in endothelial, lymphatic endothelial, and breast cancer cell migration assays. Serum from mice that were treated with 10 mg/kg morphine for 3 d displayed reduced chemotactic potential for endothelial and breast cancer cells, and elicited reduced cancer cell invasion through reconstituted basement membrane compared with serum from saline controls. This was associated with decreased circulating matrix metalloproteinase 9 (MMP-9) and increased circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3/4 as assessed by zymography and reverse zymography. By using quantitative RT-PCR, we confirmed morphine-induced alterations in MMP-9 and TIMP expression and identified organs, including the liver and spleen, in which these changes originated. Pharmacologic inhibition of MMP-9 abrogated the difference in chemotactic attraction between serum from saline-treated and morphine-treated mice, which indicated that reduced proteolytic ability mediated the decreased migration toward serum from morphine-treated mice. This novel mechanism may enable morphine administration to promote an environment that is less conducive to tumor growth, invasion, and metastasis.-Xie, N., Khabbazi, S., Nassar, Z. D., Gregory, K., Vithanage, T., Anand-Apte, B., Cabot, P. J., Sturgess, D., Shaw, P. N., Parat, M.-O. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Therapeutic vaccine to cure large mouse hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Here we report the development of a therapeutic vaccination regimen (shortened as ‘TheraVac’) consisting of intratumoral delivery of high-mobility group nucleosome-binding protein 1 (HMGN1), R848/resiquimod, and one of the checkpoint inhibitors (e.g. anti-CTLA4, anti-PD-L1, or low dose of Cytoxan). C57BL/6 mice harboring large (approximately 1 cm in diameter) established subcutaneous Hepa1-6 hepatomas were cured by intratumoral injections of TheraVac and became tumor-free long-term survivors. Importantly, the resultant tumor-free mice were resistant to re-challenge with Hepa1-6 hepatoma, not B16 melanoma, demonstrating the acquisition of hepatoma-specific immunity in the absence of any administered tumor antigen. Mechanistic studies showed that upon treatment with TheraVac, Hepa1-6-bearing mice generated increased Hepa1-6-specific CTLs in the draining lymph nodes and showed greatly upregulated expression of CXCL9, CXCL10, and IFN-γ and elevated infiltration of T lymphocytes in tumor tissues. Treatment of large Hepa1-6 hepatomas on one mouse flank also eliminated smaller (approximately 0.5 cm in diameter) hepatomas implanted on the other flank. Thus, TheraVac has potential as a curative immunotherapeutic regimen for the treatment of human HCC.

Abstract 4614: Upregulation of memory T cell population and enhancement of Th1 response by lenvatinib potentiate antitumor activity of PD-1 signaling blockade

Abstract Introduction: Lenvatinib (LEN) selectively inhibits the kinase activity of VEGFR1-3, FGFR1-4, KIT, PDGFRα, and RET, which are involved in tumor angiogenesis and tumor cell proliferation in several cancer types. Currently, Phase 1b/2 clinical trials of the combination of LEN and pembrolizumab (a monoclonal antibody [mAb] that blocks the interaction between PD-1 and its ligands) are ongoing for selected types of cancer including renal cell carcinoma, melanoma and non-small cell lung carcinoma. We have reported that tumor associated macrophage and regulatory T cell population were downregulated by treatment of LEN. In this study, mechanism of LEN and PD-1 mAb combination was investigated by flow cytometory and RNA-seq analyses. Experimental Procedure: We examined antitumor activity of combination treatment of LEN (10mg/kg, qd) and anti-mouse PD-1 mAb (500μg/mouse, twice weekly) in Ct26 and B16F10 syngeneic mouse models. In order to investigate tumor-specific memory T cell response, re-inoculation of fresh CT26 cells into tumor-free mice was conducted. For immune cell population analyses, tumor or spleen samples were analyzed by flow cytometry. Changes of molecular immune response in tumors were examined by RNAseq followed by weighted gene co-expression network analysis (WGCNA). Summary of data: Combination of LEN with PD-1 mAb showed more potent anti-tumor growth activity in CT26 and B16F10 models compared with either treatment alone. Notably, complete tumor regression was detected in a portion of mice that received combination treatment only. Re-inoculation of fresh CT26 cells into these tumor-free mice did not grow tumor; in contrast, all naïve animals grew tumor aggressively in the same experimental condition. RNAseq analysis of treated CT26 tumors indicated that combination of LEN with PD-1 mAb synergistically up-regulates IFN signaling-related genes. Flow cytometory analysis revealed that LEN increased memory T cell population compared with vehicle treatment. Conclusions: The results indicate that the combination of LEN with PD-1 mAb was more effective than either single agent alone in multiple syngeneic tumor models and was accompanied with a potent antitumor immune response, especially the up-regulation of memory T cell and Th1 activation by LEN. Citation Format: Yu Kato. Upregulation of memory T cell population and enhancement of Th1 response by lenvatinib potentiate antitumor activity of PD-1 signaling blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4614. doi:10.1158/1538-7445.AM2017-4614

Abstract 3647: Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models

Abstract Recent success of immune checkpoint inhibitors (CPIs) in the clinic underscores the role of cancer immune surveillance and the therapeutic potential of targeting immune receptors. Unlike CPIs, many members of the TNF receptor superfamily (TNFRSF), e.g., CD40, 4-1BB, OX-40, and GITR deliver agonistic signals to immune cells that may directly activate antitumor immune responses and, in combination with CPIs, may improve outcomes of active immunotherapy. Sugar-Engineered Antibody (SEA)-CD40 is a non-fucosylated, humanized IgG1 anti-human CD40 monoclonal antibody currently being evaluated in a phase 1 clinical trial in solid tumors and lymphoma. It binds FcγR3A with higher affinity and demonstrates enhanced immune stimulatory activity compared to its parent dacetuzumab. An SEA version of an anti-mouse CD40 antibody (mouse IgG2a), SEA-1C10, has been generated. As expected, SEA-1C10 bound to mouse FcγRIV, the functional homolog of human FcγR3A in mice, at a higher affinity than parent 1C10. Enhanced FcγRIV engagement by SEA-1C10 translated into stronger antitumor activity in the CD40 (-) B16F10 melanoma and CD40 (+) A20 lymphoma models. In the A20 model, tumor free mice that survived the initial A20 tumor implantation and SEA-1C10 treatment were re-challenged with live A20 cells. Twenty nine out of thirty one such tumor free mice rejected A20 tumor re-engraftment, suggesting that a strong memory immune response was elicited by SEA-1C10 treatment. The SEA-1C10/anti PD-1 combination also showed greater antitumor activity compared to SEA-1C10 or anti-PD-1 alone, supporting the hypothesis that stimulation of antigen-presenting cells by agonistic CD40 signaling can complement immune checkpoint inhibition. In addition, a transgenic (TG) mouse strain has been generated in which the endogenous Cd40 locus is inactivated by the targeted insertion of a human-mouse chimeric CD40 gene. This allows for incorporation of the extracellular domain of human CD40 while keeping the transmembrane and cytoplasmic domains of mouse CD40. Lineage-restricted expression of the CD40 TG on B cells and monocytes was confirmed. CD40 TG cells responded to SEACD40-mediated agonistic signaling in vitro by secreting CD40 signature cytokines and up-regulating the costimulatory molecules CD86 and MHC class II. Treatment of CD40 TG animals with SEA-CD40 resulted in cytokine induction and B-cell depletion. Similar to studies in human PBMCs and non-human primates, SEA-CD40 induced more robust cytokine production and immune cell activation than its fucosylated parent dacetuzumab. These experimental systems will be further applied to address clinically important questions including mechanism(s) of anti-tumor effects, biomarkers indicating immune activation, dosing strategies, and timing of combinatorial regimens with CPIs. Citation Format: Shyra J. Gardai, Weiping Zeng, Che-Leung Law. Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2017-3647

Abstract 5659: Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer

Abstract Pancreatic cancer remains a serious unmet medical need. The majority of pancreatic tumors are composed of stromal cells that make tumors resistant to conventional therapies. We are developing an approach in which a TLR9 agonist, IMO-2125, is administered intratumorally, which induces local Th1 responses and modulates the tumor microenvironment. This approach has shown promising antitumor activity in preclinical models of multiple tumor types in combination with checkpoint inhibitors. We are employing a similar approach to the treatment of pancreatic cancer. We evaluated the local therapeutic effect of IMO-2125 in a murine syngeneic pancreatic ductal adenocarcinoma (Panc02) cancer models. In the first study, C57BL/6 mice were intraperitoneally (i.p.) implanted with Panc02 cells to generate peritoneal metastatic cancer. Tumor-bearing mice (n=10 per group) were then treated with 2.5 mg/kg IMO-2125 either by i.p. or subcutaneous (s.c.) injections for six times in two weeks. In the second study, to establish dual solid tumors in C57BL/6 mice, Panc02 cells were subcutaneously implanted in both the right and left flanks of the animals. When tumors reached to 50-150 mm3, we started the intratumoral (i.t.) administration of IMO-2125 in right tumors at doses of either 0.25 mg/kg, 1 mg/kg or 2.5 mg/kg, or PBS (n=10 per group), 3x/week for three weeks. In the peritoneal metastatic pancreatic tumor model, 80% of IMO-2125 intraperitoneally treated mice survived for more than 88 days, while 90% of the control group died within 29 days. Compared to i.p. treatment, the s.c. treated group survived for only 36 days. Tumor-free mice from the IMO-2125 i.p treatment group (n=7) rejected the same tumor rechallenge, suggesting establishment of durable tumor-specific immune response. In the dual solid pancreatic tumor model, intratumoral IMO-2125 treatment led to a dose dependent antitumor activity in both injected and distant tumors. Tumors treated with 0.25, 1 and 2.5 mg/kg doses of IMO-2125 showed reductions in tumor volume by 66% (p = 0.004), 74% (p = 0.002) and 95% (p = 0.003), respectively. Moreover, the distant tumors exhibited a reduction of 0% (p = 0.65), 64% (p = 0.006) and 69% (p = 0.001), respectively. Antitumor activity was associated with increased T-cell infiltration and upregulation of the gene expression of various checkpoints in both treated and distant tumors. In an independent study, treatment with a control IMO showed minimal antitumor activity. All treatments were well tolerated. In summary, local treatment with IMO-2125 exerts antitumor activity in preclinical pancreatic cancer models, associated with an increase in T-cell infiltration in the tumor microenvironment, providing a novel approach to the treatment of pancreatic cancer. The clinical trial of intratumoral IMO-2125 is in progress in patients with melanoma. Citation Format: Daqing Wang, Evren Kocabas Argon, Fugang Zhu, Sudhir Agarwal. Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5659. doi:10.1158/1538-7445.AM2017-5659

Abstract 5599: Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy

Abstract Immune-checkpoint inhibitors are now approved for the treatment of early- and late-stage cancers. These include agents that block the T-cell regulatory protein programmed cell death 1 (PD-1) from being activated by the PD-1 ligand 1 (PD-L1) expressed on cancer cells. There is an urgent need to identify biomarkers of PD-1 pathway inhibition that would predict patient populations responsive to treatment and/or serve as surrogates for drug activity and resistance. PD-L1 expression on tumors is currently a biomarker candidate, but reliable detection and quantification methodologies have proven challenging to standardize. Recently, a soluble PD-L1 (sPD-L1) fragment was identified that can derive from cell-bound PD-L1. Retrospective clinical examinations of sPD-L1 levels in cancer patients suggest a potential use as a surrogate for disease progression and response to treatment; but few preclinical studies have been performed to test this predictive value. We undertook experiments to evaluate plasma sPD-L1 in mouse tumor models during localized primary tumor growth (after orthotopic cell implantation) and spontaneous metastatic disease progression (after surgical removal of the primary). Mouse syngeneic and human xenograft implantation models included breast, kidney, colon, and melanoma cell systems. Our results show that circulating plasma sPD-L1 can correlate with primary and metastatic progression in a stage and model specific manner. Next, we evaluated sPD-L1 following treatment with neutralizing antibodies to PD-1 and PD-L1 in tumor-free mice and found significant dose-dependent sPD-L1 increases, suggesting systemic changes may have utility as a measurement of target saturation and dosing independent of tumor growth. Finally, with current approvals of PD-1 inhibitors in renal cell carcinoma (RCC) patients previously treated with antiangiogenic agents that block vascular endothelial growth factor (VEGF), we evaluated plasma in mouse models of sunitinib resistance - a VEGF receptor tyrosine kinase inhibitor (RTKI). Our results demonstrate that VEGF pathway resistance yields changes in sPD-L1 and may be useful in predicting response to PD-1 pathway inhibition in the refractory setting. Together, these investigations suggest that circulating sPD-L1 changes during disease progression (both local and disseminated) may serve as a potential predictive biomarker for immune-checkpoint and antiangiogenic therapy. Citation Format: Michalis Mastri, Amanda Tracz, Yuhao Shi, Georg Bjarnason, Tran Nguyen, Brian Rini, John M.L. Ebos. Soluble PD-L1 as a surrogate biomarker of metastatic progression and resistance to antiangiogenic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5599. doi:10.1158/1538-7445.AM2017-5599

Abstract 3980: Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells

Abstract A heterogeneous group of immature myeloid cells which suppress the immune system are termed myeloid derived suppressor cells (MDSC). Collectively a MDSC population is comprised of monocyte-like MDSC and polymorphonuclear MDSC (PMN-MDSC). MDSC are increased in the presence of a number of different tumors. We previously demonstrated that PMN-MDSC represent the majority of MDSC in tumor-bearing mice. In a mouse model of multiple myeloma (MM) depletion of myeloid cells in vivo results in increased survival. Direct co-culture of neutrophils (CD11b+Gr1+ cells from tumor-free mice) or PMN-MDSC (CD11b+Gr1+ cells from tumor-bearing mice) and MM cells increases the survival of the MM cells from chemotherapeutic-induced death. Cell-cell contact is not necessary for the protective effect. Neutrophil and PMN-MDSC supernatants protect MM cells from cell death, suggesting the factor protecting tumor cells is soluble. Results in the mouse were confirmed using human MM cells and replicated on breast cancer cells with neutrophils or PMN-MDSC. Several cytokines and growth factors, such as IL-6 and fibroblast growth factor, are implicated in chemoresistance. The role of these neutrophil and PMN-MDSC soluble factors and the identification of novel contributors to chemoresistance are under investigation. Citation Format: Sarah Herlihy, Cindy Lin, Maria Ozeriva, Alfred Garfall, Dan Vogl, Dmitry Gabrilovich, Yulia Nefedova. Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2017-3980

Abstract 4039: Riluzole synergizes with paclitaxel to induce apoptosis and inhibit cell growth in triple negative breast cancer

Abstract Introduction: Systemic treatment for triple negative breast cancer (TNBC) includes paclitaxel, which works by inhibiting the breakdown of microtubules. We previously observed that riluzole, an FDA-approved drug for the treatment of amyotrophic lateral sclerosis, can inhibit TNBC proliferation, invasion, and colony formation. We now test whether riluzole can act synergistically with paclitaxel to inhibit TNBC growth and induce apoptosis in TNBC. Methods: After treatment of various TNBC cell lines with constant ratio concentrations of riluzole and paclitaxel we conducted cell proliferation transformation assays (MTT) to measure cell inhibition potential. Synergy or additivity was determined by the Chou-Talalay using Compusyn software. PARP cleavage normalized to GAPDH was also measured by Western blot to estimate apoptosis due to the riluzole-paclitaxel combination. In vivo synergy was studied using a xenograft study with MDA-MB-231, a human TNBC cell line sensitive to riluzole. Results: MTT results show that riluzole and paclitaxel work synergistically to inhibit cell proliferation in all TNBC cell lines tested, as determined by isobolograms and CI values <1. The strongest synergistic effect was observed in the cell lines SUM159, SUM149, and SUM229, where synergism occurred at Fa values as low as 0.5 (50% inhibition of cell proliferation). Measuring PARP cleavage, we demonstrated that the riluzole-paclitaxel drug combination induces greater apoptosis than does either drug alone. The significant impact of the drug combination was best demonstrated in cell lines SUM159, SUM149 and SUM229, but was observed in all cell lines tested. In the xenograft study, the highest non-toxic dose of paclitaxel (7.2 mg/kg) combined with riluzole resulted in 8/9 tumor-free mice, with a tumor growth inhibition ratio (T/C) of 0%, where <10% is considered highly therapeutic. This result was better than riluzole monotherapy (0/9 tumor-free and T/C = 99%) or paclitaxel monotherapy (4/8 tumor-free and T/C = 8%). Conclusions: Our results demonstrate that the addition of riluzole to paclitaxel results in at least additive and in many cases synergistic effects against TNBC. Our observations thus suggest that repurposing riluzole, which is an oral drug with an excellent safety profile, to add to paclitaxel to treat TNBC represents a potential strategy to improve the efficacy of adjuvant and neoadjuvant treatments for TNBC and/or reduce chemotherapy doses and toxicity in patients. Our data support the need to proceed to clinical trials to test this approach in patients. Citation Format: Miriam A. Bukhsh, Cecilia L. Speyer, Waris S. Jafry, Rachel E. Sexton, David Thomas, David Gorki. Riluzole synergizes with paclitaxel to induce apoptosis and inhibit cell growth in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4039. doi:10.1158/1538-7445.AM2017-4039

Abstract 1041: Binding of soluble DC-HIL to endothelial cell creates immunosuppression on premetastatic niches

Abstract Highly metastatic attribute of melanoma can be recapitulated in a mouse model by high potential of B16 melanoma-derived soluble factors that form more premetastatic niches (PMNs) in distal organs than those of LL2 lung carcinoma. Having shown that the T cell-inhibitory receptor DC-HIL is a potent promoter of B16 melanoma growth and that B16 cells secrete it into blood circulation as a soluble receptor (sDC-HIL), we hypothesized that sDC-HIL accounts for the metastatic nature. We tracked sDC-HIL in various organs of melanoma-bearing mice by immunoblotting and showed that sDC-HIL was expressed in particular organs; at highest levels in bone marrow (BM) and lung; and no-to-little in lymphoid organs and liver. We next examined effects of sDC-HIL on LL2 cells (no DC-HIL expression). In a model of spontaneous metastasis quantified by colonogenic assay, syngeneic mice implanted subcutaneously with LL2- transfected-sDC-HIL cells produced 6-fold more lung mets than mice with LL2-transfected-GFP. We obtained similar results in a model of experimental metastasis (EM), in which LL2 cells injected i.v. in tumor-bearing mice. To establish specificity, similar mice were treated i.v. with anti-DC-HIL mAb 2 h before and 24 h after infusion of LL2 cells: The mAb led to 80% reduction in lung mets in mice with sDC-HIL-LL2 tumor. Next we infected tumor-free mice with lentiviruses for sDC-HIL/GFP or GFP gene and then performed the EM assay: lung mets in mice expressing sDC-HIL were 5-fold greater than control. To determine whether sDC-HIL is responsible for the ability of B16-conditioned media (CM) to induce metastasis, tumor-free mice were injected i.p. daily with CM from LL2, B16, or DC-HIL-knocked down (KD) B16 cells and then assayed for EM. B16-CM induced significantly more lung mets than LL2-CM, with mets almost corresponding to that produced by KD-B16-CM. We tracked the fate of sDC-HIL in lung by immunofluorescence and showed sDC-HIL accumulated on particular (not all) endothelial cells (EC), at sites where LL2 and BM-cells migrated. Kinetic studies in BM-chimera mice showed that sDC-HIL arrives at lung sites before VEGFR1+ BM-progenitors, which initiate formation of PMNs. Since the mets-enhancing effect of sDC-HIL was not seen in immunodeficient mice, we asked whether lungs expressing sDC-HIL were immunosuppressive. CFSE-labeled CD8+ T cells were infused into mice with sDC-HIL- or GFP-LL2 tumors, and their proliferation assayed by FACS: T-cell proliferation in lungs expressing sDC-HIL was reduced significantly (28%) vs. control (81%). Because sDC-HIL by itself is not suppressive, we asked whether binding of sDC-HIL by EC makes immunosuppressive. CD8+ T cells were co-cultured with sDC-HIL-bound EC and then co-stimulated: sDC-HIL-bound EC completely suppressed the IFN-γ response. Thus, sDC-HIL critically contributes to the highly metastatic potential by making PMNs immunosuppressive even before the arrival of tumor cells. Citation Format: Vijay Ramani, Takahiro Teshima, Kyoichi Tamura, Jin-Sung Chung, Ponciano Cruz, Kiyoshi Ariizumi. Binding of soluble DC-HIL to endothelial cell creates immunosuppression on premetastatic niches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2017-1041

Abstract 621: Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models

Abstract TUSC2, a pro-apoptotic tumor suppressor gene whose expression is lost or decreased in most lung cancers, activates the innate immune system through initiation of broad spectrum cytokine secretion and natural killer (NK) cell activation. TUSC2 delivered systemically by nanovesicles has mediated tumor regression in metastatic non-small cell lung cancer clinical trials. We studied the effect of TUSC2 on immune cell populations and the anti-tumor activity of TUSC2 in combination with anti-PD1 checkpoint blockade in two syngeneic mouse models: C57BL/6 mice subcutaneously injected with murine lung adenocarcinoma cell line CMT/167-luc cells (KrasG12V mutation) and 344SQ (KrasG12D allele and a knock-in Trp53R172HΔG allele) adenocarcinomas which metastasize to the lung in 129S2 mice. Tumor growth was monitored by scoring ex-vivo luminescence using the IVIS Imaging System 200. Multi-color flow cytometry was used for immune profiling of circulating immune cells after nanovesicle mediated TUSC2 intravenous injection. Cytokine gene expression in response to TUSC2 in sorted immune subpopulations was determined by real-time PCR. Tumor growth was significantly reduced with TUSC2 treatment compared with no treatment in both subcutaneous and metastatic mouse models. Synergistic anti-tumor activity was observed when TUSC2 was combined with anti-PD1 verified in five independent experiments. In the lung metastasis model, mice treated with TUSC2 + anti-PD1 lived significantly longer than with single agent treatment. Circulating NK cells increased three fold following TUSC2 nanovesicle intravenous injection both in tumor free and tumor bearing mice which correlated with tumor regression and survival. Cytotoxic T lymphocyte responses were increased whereas Tregs and MDSCs decreased with TUSC2 alone and TUSC2+anti-PD1 treatment. The levels of T cell checkpoint markers PD1, CTLA-4, LAG-3, and TIM-3 evaluated by flow cytometry were decreased after TUSC2 treatment. TUSC2 anti-tumor response was abolished when NK cells were depleted indicating NK cells are important mediators of the TUSC2 treatment effect. Single cell suspension analysis by flow cytometry showed high numbers of NK cells infiltrating lung tumor metastases after TUSC2 treatment. The number of tumor nodules in the lung was significantly less following treatment with TUSC2 nanovesicles compared with control. IL-15 gene expression which mediates NK cell proliferation, was increased by TUSC2. In conclusion, systemic TUSC2 nanovesicle immunogene therapy combined with checkpoint blockade showed synergistic anti-tumor efficacy and activated the immune system through upregulation of NK cells and CTL and downregulation of regulatory cells. Citation Format: Ismail M. Meraz, Mourad Majidi, RuPing Shao, Meng Feng, Xiaobo Cao, David Rice, Boris Sepesi, Lin Ji, Jack Roth. Tumor suppressor TUSC2 immunogene therapy is synergistic with anti-PD1 in lung cancer syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 621. doi:10.1158/1538-7445.AM2017-621

Abstract 1602: Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance

Abstract Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to a family encompassing 6 interleukins (IL-17A to F) and binds to the IL-17 receptor B (IL-17RB). Recently, amplified IL-17B/IL-17RB signaling was found critical for breast and pancreatic tumorigenesis and elevated expression of IL-17RB has been associated with the shortest survival rates in patients with breast or pancreatic cancer. Using IL-17B knock-out (IL-17B KO) mice we demonstrate here that melanoma, fibrosarcoma and breast cancer cell tumorigenicity is strongly impaired in immunocompetent IL-17B KO mice compared to WT littermates, including a large number of tumor free mice. Reduced tumor incidence in IL-17B KO mice is associated with alterations of the immune tumor microenvironment especially within innate lymphocyte and myeloid sub-populations. We further demonstrate that IL-17B is a key cytokine shaping the tumor initiating cancer cell niche. Indeed, MDA-MB-468 human breast cancer cells overexpressing IL-17B exhibit 10 times higher frequency of tumor initiating cells when xenografted at a serial limiting dilution in nude mice. Tumor progression is, again, associated with alterations of NK cells within the tumor microenvironment and with increased percentages of CD44hi/CD24lo tumor cells, a phenotype associated with breast cancer stem cells (CSC). This is associated with resistance to conventional chemotherapeutic agents such as taxol, an effect that is totally abrogated by disrupting IL-17B-IL-17RB signaling with a neutralizing antibody. Altogether our results point out the key role of IL-17B in regulating the immune microenvironment as well as cardinal features of CSC, one of the alleged causes of resistance to therapy and tumor relapse. Thereby, IL-17B and its receptor appear as potential therapeutic targets for cancer immunotherapy. Collectively, these data support the ongoing development of IL-17B neutralizing antibodies. Citation Format: Emilie Laprevotte, Aurélie Docquier, Jeremy Bastid, Cécile Déjou, Marion Lapierre, Gilles Alberici, Armand Bensussan, Jean-François Eliaou, Nathalie Bonnefoy. Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2017-1602