Syngeneic Immunocompetent Mouse Model Research Articles

Abstract B78: Irreversible electroporation (IRE) acts as an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer

Abstract Introduction: Cancer immunotherapy has made waves of progress in recent years with the advent of checkpoint inhibitor therapies against several cancers. However, pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded immunotherapy approaches. PC hosts a notoriously immunosuppressive microenvironment comprised of an abundance of immunosuppressive tumor associated macrophages, myeloid derived suppressor cells (MDSCs) and regulatory T-cells with a scarcity of effector CD8+ T-cells. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures. Unlike thermal ablation techniques, IRE results in a gradual apoptotic cell death and is currently in clinical use for selected patients with locally advanced PC. We hypothesize that the abundant release of antigens by IRE can induce an “in situ vaccination” effect that can elicit adaptive T-cell-mediated anti-tumor immune responses. Methods: We have developed a robust syngeneic immunocompetent mouse model of PC using a cell line established from a tumor arising in a LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC). We have utilized the ECM 830 square wave pulse generator to deliver IRE (100 μsec pulses of electricity at 1500 V/cm using a two-needle array probe) to subcutaneous tumors measuring 5-7 mm in diameter. The effects of IRE on primary tumor growth and secondary tumor challenge were observed. Flow cytometry-based immunoprofiling studies of tumors at one week post-IRE were performed in triplicate and presented as mean difference between IRE and no treatment (standard error). *P<0.01 by student’s t-test was considered statistically significant. Results: Vaccination studies with irradiated KPC cells confirmed that they were immunogenic in C57BL/6 mice. In preliminary studies, we identified the minimal IRE dose to induce tumor regression to be 150 pulses, with complete regression in 3 of 9 tumors (33%). Survival was prolonged in the IRE groups, particularly in mice with complete regression. This outcome was confirmed to be immune-mediated, as IRE of tumor-bearing immunodeficient Rag-1-/- mice, which lack functional T and B cell components, results in progressive outgrowth with similar kinetics as untreated mice. When immunocompetent mice with tumor regression were re-challenged two weeks later with 105 cells injected into the contralateral flank, we observed no growth in the secondary tumors, confirming adaptive immune activation post-IRE. Tumor immunoprofiling revealed a 43±2%* decrease in the number of tumor-infiltrating MDSCs (CD11b+/CD11c+/Gr-1hi) post-IRE, with a similar decrease in the respective tumor draining inguinal lymph nodes. An increase in total tumor infiltrating lymphocytes (TILs) was also observed, including an 18±2%* increase in CD8+ TILs and a concomitant large increase (2.5 fold*) in PD-1+/CD8+ double positive T-cells. Conclusion: These results suggest that IRE is capable of triggering an antitumor immune response that contributes to the local effects of IRE and inhibits growth of secondary tumor re-challenge. IRE affects the composition of immune cells in the tumor microenvironment, including a large increase in PD-1+/CD8+ TILs. These effects may be augmented by combining IRE with checkpoint blockade, which is a focus of our ongoing studies. Citation Format: Jayanth Shankara Narayanan, Tomoko Hayashi, Aaron M. Miller, Stephen P. Schoenberger, Rebekah R. White. Irreversible electroporation (IRE) acts as an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B78.

BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer

BackgroundAngiogenesis has become an attractive target for cancer therapy. However, despite the initial success of anti-VEGF (Vascular endothelial growth factor) therapies, the overall survival appears only modestly improved and resistance to therapy often develops. Other anti-angiogenic targets are thus urgently needed. The predominant expression of the type I BMP (bone morphogenetic protein) receptor ALK1 (activin receptor-like kinase 1) in endothelial cells makes it an attractive target, and phase I/II trials are currently being conducted. ALK1 binds with strong affinity to two ligands that belong to the TGF-ß family, BMP9 and BMP10. In the present work, we addressed their specific roles in tumor angiogenesis, cancer development and metastasis in a mammary cancer model.MethodsFor this, we used knockout (KO) mice for BMP9 (constitutive Gdf2-deficient), for BMP10 (inducible Bmp10-deficient) and double KO mice (Gdf2 and Bmp10) in a syngeneic immunocompetent orthotopic mouse model of spontaneous metastatic breast cancer (E0771).ResultsOur studies demonstrate a specific role for BMP9 in the E0771 mammary carcinoma model. Gdf2 deletion increased tumor growth while inhibiting vessel maturation and tumor perfusion. Gdf2 deletion also increased the number and the mean size of lung metastases. On the other hand, Bmp10 deletion did not significantly affect the E0771 mammary model and the double deletion (Gdf2 and Bmp10) did not lead to a stronger phenotype than the single Gdf2 deletion.ConclusionsAltogether, our data show that in a tumor environment BMP9 and BMP10 play different roles and thus blocking their shared receptor ALK1 is maybe not appropriate. Indeed, BMP9, but not BMP10, acts as a quiescence factor on tumor growth, lung metastasis and vessel normalization. Our results also support that activating rather than blocking the BMP9 pathway could be a new strategy for tumor vessel normalization in order to treat breast cancer.

Abstract 1698: Loss of MHC-I expression is associated with altered immune microenvironment and decreased response to PD-1 checkpoint inhibition

Abstract Cancer immunotherapies, such as PD-1 inhibitors, achieve durable responses in a subset of cancer patients, however, a significant proportion of patients fail to respond to treatment. Defects in antigen presentation, such as the loss of MHC-I expression, are a major mechanism of resistance to PD-1 inhibitors. Developing new therapeutic strategies that overcome the lack of antigen presentation will improve cancer treatment and patient survival. To develop a pre-clinical model to evaluate therapies that could overcome resistance, we have generated a unique PD-1 blockade sensitive, syngeneic immunocompetent melanoma mouse model. We generated defects in antigen presentation using the CRISPR/Cas9 system, creating B2m-deficient melanoma cell lines. We demonstrated that B2m is necessary for IFN-gamma induced MHC-I surface expression. Unlike control tumors, we found that B2m-deficient tumors were resistant to PD-1 blockade, leading to decreased mouse overall survival. Using multi-parameter flow cytometry, we showed an increase in NK cell, regulatory T cell and dendritic cell infiltration in B2m-deficient tumors compared to controls, while the other immune cell population were unchanged. Since NK cell activity is negatively regulated by MHC-I, our findings suggest that additional factors are required to further invigorate infiltrating NK cells and to trigger a NK cell-mediated anti-tumor response. We are currently evaluating new approaches targeting NK cells and regulatory T cells on B2m-deficient tumors. This pre-clinical model could enable the development of a therapeutic strategy serving to improve the treatment of tumors lacking antigen presentation. Citation Format: Cecile Gstalder, Priya Pancholi, Dorota Sadowicz, Rizwan Haq. Loss of MHC-I expression is associated with altered immune microenvironment and decreased response to PD-1 checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1698.

T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression

The discovery of genes and molecular pathways involved in the formation of brain metastasis would direct the development of therapeutic strategies to prevent this deadly complication of cancer. By comparing gene expression profiles of Estrogen Receptor negative (ER-) primary breast tumors between patients who developed metastasis to brain and to organs other than brain, we found that T lymphocytes promote the formation of brain metastases. To functionally test the ability of T cells to promote brain metastasis, we used an in vitro blood–brain barrier (BBB) model. By co-culturing T lymphocytes with breast cancer cells, we confirmed that T cells increase the ability of breast cancer cells to cross the BBB. Proteomics analysis of the tumor cells revealed Guanylate-Binding Protein 1 (GBP1) as a key T lymphocyte-induced protein that enables breast cancer cells to cross the BBB. The GBP1 gene appeared to be up-regulated in breast cancer of patients who developed brain metastasis. Silencing of GBP1 reduced the ability of breast cancer cells to cross the in vitro BBB model. In addition, the findings were confirmed in vivo in an immunocompetent syngeneic mouse model. Co-culturing of ErbB2 tumor cells with activated T cells induced a significant increase in Gbp1 expression by the cancer cells. Intracardial inoculation of the co-cultured tumor cells resulted in preferential seeding to brain. Moreover, intracerebral outgrowth of the tumor cells was demonstrated. The findings point to a role of T cells in the formation of brain metastases in ER- breast cancers, and provide potential targets for intervention to prevent the development of cerebral metastases.

Abstract 124: Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy

Abstract ONC201, a first-in-class oral anti-tumor agent, upregulates the pro-apoptotic immune cytokine TRAIL and activates the integrated stress response leading to upregulation of death receptor 5 in bulk tumor and cancer stem cells. We previously demonstrated that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo while suppressing Akt/ERK signaling in tumors in a dose/frequency-dependent manner (Wagner et al., AACR, 2016). We also provided evidence that ONC201 exhibits a potent anti-metastatic effect (Wagner et al., AACR, 2016). We observe accumulation and activation of TRAIL-secreting NK+ cells within ONC201-treated tumors in C57/BL6, Balb/c, and athymic nude tumor-bearing mice. Importantly, ONC201 exerts in vivo anti-tumor efficacy on tumor cell lines that are ONC201-resistant in vitro, including acquired stable resistance. Using the NK-depleting antibody GM1, we demonstrate that the activation and TRAIL secretion of NK cells by ONC201 significantly contributes to in vivo anti-tumor efficacy, including TRAIL/ONC201-resistant tumors. We are currently investigating how ONC201 recruits NK cells to the tumor by examining NK-recruiting chemokine factors within the tumor site. We have also demonstrated upregulation of CD3+ T cells by ONC201 in syngeneic mice. Finally, we observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients upon ONC201 administration in the clinic. Our results demonstrate novel and potentially significant increases in cytotoxic NK cell recruitment to tumors. The results offer a unique pathway of immune stimulation for cancer therapy that may be combined with immune checkpoint or targeted cancer therapy strategies. We are currently investigating the role of NK cells and CD3+ cells in ONC201’s ability to inhibit metastasis by using a metastatic model that involves surgically removing the primary tumor and allowing metastases to grow in vivo before treatment. These findings indicate that ONC201 possess immunomodulatory activity and provide a rationale for combining ONC201 with PD-1/PDL-1 inhibitors, a combination we are currently testing in syngeneic immunocompetent mouse models. Citation Format: Jessica Wagner, C. Leah Kline, Lanlan Zhou, Andrew Zloza, Charles Chesson, Jenna Newman, Howard Kaufman, Joseph Bertino, Mark Stein, Wafik El-Deiry. Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy [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 124. doi:10.1158/1538-7445.AM2017-124

Converting Lymphoma Cells into Potent Antigen-Presenting Cells for Interferon-Induced Tumor Regression.

Anti-hCD20 is a therapeutic mAb that is clinically used to treat B-cell lymphoma. Some lymphomas are resistant to anti-hCD20; others relapse after treatment with anti-hCD20. Using a syngeneic immunocompetent mouse model, we observed that targeting lymphoma with interferon-α (IFNα) abolished resistance of B-cell lymphoma to anti-CD20 while limiting interferon (IFN)-associated systemic toxicity in the host. Control of tumors by a fusion of anti-CD20 and IFNα (anti-CD20-IFNα) depended on existing tumor-infiltrating CD8+ T cells. Although lymphomas were resistant to IFN-directed killing, IFN-exposed tumor cells became the dominant antigen-presenting cells (APC) for the reactivation of tumor-infiltrating CD8+ T cells that then controlled those lymphomas. Anti-CD20-IFNα also abolished checkpoint blockade resistance in advanced B-cell lymphoma. Our findings indicate that anti-CD20-IFNα eradicates B-cell lymphoma by employing tumor cells as APCs to reactivate tumor-infiltrating CD8+ T cells and synergizing with anti-PD-L1 treatment. Cancer Immunol Res; 5(7); 560-70. ©2017 AACR.

Abstract 2938: Tumour-associated macrophage polarisation and re-education with oncolytic viruses

Abstract Tumor-associated macrophages (TAMs) promote key processes in tumour progression through their plasticity and capacity to become polarised by tumours to have an immunosuppressive phenotype. However, the plasticity of these cells provides an opportunity for therapeutic ‘reprogramming’ towards a classically activated inflammatory phenotype thus preventing tumour-associated immunosuppression and inducing anti-tumour immunity. Oncolytic viruses (OV) represent a new class of anti-cancer therapeutics that promote anti-tumour responses through selective tumour cell killing and the induction of systemic anti-tumour immunity. We propose that TAMs can be targeted using an OV, leading to altered macrophage polarization and a reduction in tumour growth and metastasis. Experimental Design: In-vitro, cytotoxicity, viral replication and cytokine expression was assessed in a panel of primary human and murine macrophages following exposure to Sephrevir, an ICP34.5-deleted oncolytic herpes-simplex virus currently in Phase 1/2a trials. In-vivo, murine TS1-PyMT cells were implanted into the mammary fat pads of female FVB mice to establish a syngeneic immunocompetent mouse model of breast cancer. Tumour growth and overall survival were assessed following administration of Sephrevir given either by intratumoural (I.T) or intravenous injection (I.V). Immune cell enumeration and protective immunity were assessed post-mortem by immunofluorescence and flow cytometry. Results: Both primary human and mouse macrophages served as a host for oncolytic viral replication. Sephrevir was also found to attenuate TAM phenotypes as evidenced by increased “M1” receptor signatures (e.g. CD40, CD80, CD86, MHC II), increased pro-inflammatory gene expression (e.g. IL-1, IL-6, IL-8, CXCL10, TNF-a) and increased nitric oxide production. I.T and I.V administration of Sephrevir led to marked tumour shrinkage as well as the recruitment of cytotoxic T cells. Moreover, TAM polarisation was evident based on a change in signature with TAMs becoming skewed to IL-12hi/IL-10lo and a change in the iNOS/arginase1 ratio. Conclusions: Our results demonstrate that Sephrevir is associated with in vivo anti-tumour effects and importantly can be used for manipulating the phenotype of the abundant macrophage population located within the tumour microenvironment. Citation Format: Munitta Muthana, Emer Murphy, Amy Kwan, Natalie Winder, Joe Conner. Tumour-associated macrophage polarisation and re-education with oncolytic viruses [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 2938. doi:10.1158/1538-7445.AM2017-2938

Low-dose glucocorticoids suppresses ovarian tumor growth and metastasis in an immunocompetent syngeneic mouse model.

Ovarian cancer has the highest mortality rate among gynecologic malignancies. Despite chemotherapy and surgical debulking options, ovarian cancer recurs and disseminates frequently with a poor prognosis. We previously reported a novel role of glucocorticoids (GCs) in metastatic ovarian cancer by upregulating microRNA-708. In this study, we used an immunocompetent syngeneic mouse model and further evaluated the effect and optimal dosages of GCs in treating metastatic ovarian cancer. The treatment of C57BL/6-derived ovarian cancer ID-8 cells with a synthetic GC, dexamethasone (DEX), induced the expression of microRNA-708, leading to decreased cell migration and invasion through targeting Rap1B. Administration of DEX at a low dose, as low as 5 μg/kg body weight, inhibited the primary tumor size and abdominal metastasis in mice bearing ID-8 cell-derived ovarian tumors. In the treated primary tumors, microRNA-708 was upregulated, whereas some proinflammatory cytokines, namely interleukin (IL)-1β and IL-18, were downregulated. The number of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment were reduced. Overall, our study shows that low-dose GCs can suppress ovarian cancer progression and metastasis likely through not only the upregulation of the metastasis suppressor microRNA-708, but also the modulation of TAMs and MDSCs in the tumor microenvironment.

Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.

Parity History Determines a Systemic Inflammatory Response to Spread of Ovarian Cancer in Naturally Aged Mice.

Aging intersects with reproductive senescence in women by promoting a systemic low-grade chronic inflammation that predisposes women to several diseases including ovarian cancer (OC). OC risk at menopause is significantly modified by parity records during prior fertile life. To date, the combined effects of age and parity on the systemic inflammation markers that are particularly relevant to OC initiation and progression at menopause remain largely unknown. Herein, we profiled a panel of circulating cytokines in multiparous versus virgin C57BL/6 female mice at peri-estropausal age and investigated how cytokine levels were modulated by intraperitoneal tumor induction in a syngeneic immunocompetent OC mouse model. Serum FSH, LH and TSH levels increased with age in both groups while prolactin (PRL) was lower in multiparous respect to virgin mice, a finding previously observed in parous women. Serum CCL2, IL-10, IL-5, IL-4, TNF-α, IL1-β and IL-12p70 levels increased with age irrespective of parity status, but were specifically reduced following OC tumor induction only in multiparous mice. Animals developed hemorrhagic ascites and tumor implants in the omental fat band and other intraperitoneal organs by 12 weeks after induction, with multiparous mice showing a significantly extended survival. We conclude that previous parity history counteracts aging-associated systemic inflammation possibly by reducing the immunosuppression that typically allows tumor spread. Results suggest a partial impairment of the M2 shift in tumor-associated macrophages as well as decreased stimulation of regulatory B-cells in aged mice. This long term, tumor-concurrent effect of parity on inflammation markers at menopause would be a contributing factor leading to decreased OC risk.

Abstract 4024: Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer

Abstract Background: Ovarian cancer (OC) progression is accompanied by the establishment of stable and transcriptionally repressive epigenetic modifications. An important mechanism of immune evasion is represented by epigenetic silencing of tumor antigens (NY-ESO-1, Muc16 and MAGE). We hypothesize that by reversing DNA methylation, DNA methyl transferase inhibitors (DNMTIs) restore the expression of such antigens, potentiating anti-tumor immune response. The targeting of immune checkpoints regulated by programmed cell death protein-1(PD-1) signaling represents a novel therapeutic strategy in cancer, including in OC. Here we set out to measure the anti-tumor effects of epigenetic priming in combination with PD-1/PDL-1 blockade in OC preclinical models. Methods: The ID8 intraperitoneal (ip) immunocompetent syngeneic mouse model was used to measure the effects of the novel DNMTI guadecitabine (SGI-110, Astex Pharmaceuticals Inc) and PDL1 blockade. The experimental groups consisted of non-specific IgG (control), guadecitabine 2mg/m2 sq bi-weekly, murine anti-PDL1 inhibitory antibody (10mg/kg) bi-weekly and combination of guadecitabine with anti-PDL1 antibody (n = 6 mice/group, 3 week treatment). Immune cells collected from the ascites and spleens of tumor bearing mice were either directly processed or co-cultured with ID8 cells for 48 hours. Cells were immuno-phenotyped by flow cytometry. In OC cells treated with guadecitabine, changes in gene expression were analyzed by real time-PCR. Results: In ID8 tumors bearing mice, the combination of PDL1 blockade with guadecitabine significantly decreased primary tumor formation (P<0.05) and malignant ascites accumulation (P<0.0001) compared with the control group. Treatments were well tolerated without detectable weight changes attributable to toxicity. CD8+ cell fractions expressing the exhaustion markers (PD1+ or CTL4+) isolated from the ascites and spleens of control mice were diminished by guadecitabine, PD-L1 inhibitory antibody, and the combination treatment. Guadecitabine and the combination regimen increased CD8+/MHC1+ and CD8+/CD40+ cell populations. Treatment with DNMTIs significantly increased the expression of tumor antigens Muc16, Mage A2, A11, and NY-ESO 1 (p<0.01) in several OC cell lines. Conclusions: Guadecitabine in combination with anti-PDL1 antibody induced striking anti-tumor effects in an immunocompetent OC syngeneic model by activating cytotoxic T-cells. These data support clinical strategies utilizing epigenetic priming using DNMTI in combination with immune checkpoints inhibitors. Citation Format: Yu Qing, Salvatore Condello, Katie J. Meyer, Andrea Caperell-Grant, Kenneth P. Nephew, Daniela Matei. Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer. [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 4024.

Abstract 1554: Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia

Abstract Despite recent advances and better diagnostics, the major challenge is that metastatic breast cancer is still incurable and remains leading cause of cancer related death. Cachexia is considered to be a chronic inflammatory syndrome which is defined by loss of skeletal muscle mass (with or without loss of adipose mass), negative energy and metabolic balance, and systemic inflammation. Cancer patients who develop cachexia are more susceptible to infections and sepsis. Clinical studies suggest that cachexia in cancer patients cannot be fully reversed by conventional nutritional supports, which distinguishes this condition from anorexia. Due to its complexity and lack of clinical biomarkers, currently there is no standard treatment for these patients. Therefore cachexia remains a largely underestimated and untreated condition. Nearly 60-80% of the advanced/ metastatic cancer patients experience cachexia, a condition that accounts for 20% of cancer-related deaths. Chronic inflammation has been recognized as a risk factor contributing to the etiology of many human malignancies. Accumulating evidence suggest that tumor infiltrating immune cells (mainly myeloid origin) differentiate into cells that promote tumor growth and metastasis via inducing a systemic inflammation. Our preliminary studies suggest that systemic induction and infiltration (tumor, bone marrow, spleen, liver, and lung) of myeloid derived suppressor cells (MDSC) generate a pro-inflammatory micro-environment and are a major source of inflammatory cytokines, many of which are implicated in cancer cachexia. Using a murine breast cancer in a syngeneic (immunocompetent) mouse model we show that metastatic (4T1) murine tumor produce significantly higher level of inflammatory cytokines and is able to induce systemic expansion and infiltration of MDSC compared to non-metastatic murine tumor (EMT6). Furthermore, injection of condition media from metastatic 4T1 tumor cells is also able to induce MDSC expansion in vivo suggesting that tumor-produced factors play role in this process. Moreover, we demonstrate the involvement of the inflammatory cytokines in muscle wasting as shown by co-culture experiments with C2C12 myoepithelial cells and analysed the expression of cachexia markers such as E3 ubiquitin ligases Trim63 and Fbxo3, Myh1(myosin heavy chain), Stat3 and NFkB pathway activation, and elevation of pro-cachexia cytokines. Our preliminary studies demonstrated that the monocytic MDSC induce EMT phenotype and contribute to the dissemination of tumor cells while the granulocytic MDSC promote the metastatic outgrowth, and present higher infiltration in the lungs. We therefore propose that tumor-induced inflammatory cytokines play role in induction of MDSC and further elevation of inflammatory markers leading to metastasis and cancer cachexia. Citation Format: Maria Ouzounova, EunMi Lee, Raziye Piranlioglu, Ena Novakovic, Mehmet Demirci, Sumeyye Korkaya, Alicia Hudson, Hasan Korkaya. Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia. [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 1554.

Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy.

Targeting of heme oxygenase-1 as a novel immune regulator of neuroblastoma.

Heme oxygenase (HO)-1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO-1 expression in samples from NB patients and show that targeting of HO-1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO-1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO-1 by systemic as well as tumor-specific zinc protoporphyrin-mediated HO-1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO-1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T-effector cells. This in turn reverted HO-1-dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO-1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.

Modulation of Natural Killer Cell Activity in the Setting of Oncolytic Virotherapy and with a Chimeric Antigen Receptor

Natural killer (NK) cells are innate lymphocytes that can rapidly eradicate tumor cells, especially those lacking MHC Class I molecules. NK cells can also rapidly eradicate herpes virus-infected cells. We designed an oncolytic herpes virus (oHSV) to selectively infect, replicate within, and lyse glioblastoma (GBM), a devastating brain tumor with a median survival of only 15 months following diagnosis. We have shown that the rapid influx of NK cells limits oHSV efficacy in GBM as they impede oHSV replication and spread [Alvarez-Breckenridge et al., Nat Med, 2012, 18(12):1827-34]. In the current study, we developed NK cell-based novel GBM therapies by decreasing the brain influx of NK cells to enhance the efficacy of oHSV, while arming NK cells in the brain with a chimeric antigen receptor (CAR) that targets both the wild-type EGFR and its mutant form EGFRvIII, two GBM tumor-associated antigens. We then investigated the synergistic effects between EGFR-CAR NK cells and oHSV.Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine of NK cells [Yu et al, Immunity, 2006, 24(5):575-90]. We first determined if oHSV efficacy for treatment of GBM would be augmented by inhibiting anti-oHSV activity of NK cells with TGF-β pre-treatment. In vitro, NK cells pre-treated with TGF-β displayed less cytolytic capacity against oHSV-infected GBM cell lines and patient-derived GBM stem-like cells. In viral replication assays, co-culturing oHSV-infected GBM cells with NK cells pre-treated with TGF-β significantly increased virus titers. In an immunocompetent syngeneic GBM mouse model,administration of TGF-β to GBM-bearing mice prior to oHSV injection significantly inhibited intracranial infiltration and activation of NK cells (P < 0.05). In orthotopic human GBM xenograft mouse models and in syngeneic GBM mouse models, TGF-β treatment in vivo prior to oHSV therapy resulted in inhibition of NK cell infiltration, suppression of tumor growth and significantly prolonged survival of GBM-bearing mice (P < 0.05). Furthermore, depletion of NK cells incompletely blocked the positive effects of in vivo treatment of GBM with TGF-β on survival, suggesting that TGF-β may also directly act on other innate immune cells such as macrophages/microglia. These data demonstrate a single dose of TGF-β prior to oHSV administration enhances anti-tumor efficacy for GBM at least in part through the transient inhibition of the innate immune responses to oHSV infection.We next investigated whether NK cell activity could be enhanced to more directly target brain tumors while sparing eradication of oHSV. We therefore infected both human NK-92 cells and primary human NK cells to express the second generation CAR targeting both EGFR and EGFRvIII that we designed. Further, we asked if the treatment with EGFR-CAR NK cells plus oHSV could create a therapeutic synergy for the treatment to brain tumors. In vitro, compared with mock-transduced CAR-NK-cells, EGFR-CAR NK cells exhibited significantly higher cytotoxicity and IFN-γ production when co-cultured with tumor cells, for both NK-92 and primary NK cells (P < 0.01). Further, significantly higher cytolytic activity against tumor cells was obtained when CAR NK cells were combined with oHSV-1 infection of tumor cells, compared to either of the monotherapies alone (P < 0.05). In mice, to avoid oHSV clearance by the EGFR-CAR NK cells following the inoculation of the mouse with tumor cells, we administered these two agents sequentially; administering EGFR-CAR NK cells directly into the tumor first as a single injection of 2 × 106 cells, followed by intracranial infection with 2 × 105 plaque-forming units oHSV five days later, presumably after EGFR-CAR NK survival has diminished. Compared to vehicle controls, intracranial administration of either EGFR-CAR NK cells or oHSV blunted tumor growth. However, the combination of EGFR-CAR NK cells followed by oHSV infection resulted in significantly more efficient killing of tumor cells (P < 0.05) and significantly longer survival for tumor-bearing mice when compared to either monotherapy alone.Collectively, our studies demonstrate that in animal tumor models, we can combine novel NK cell and oHSV therapies to significantly improve survival. DisclosuresNo relevant conflicts of interest to declare.

Abstract B73: Ketogenic diet enhances immunity to glioblastoma

Abstract Glioblastomas are the most common and malignant brain tumor in adults and account for over 50% of all gliomas. Current standard of care combines surgical resection with temozolomide chemotherapy and radiotherapy, but 5 year survival rates remain less than 20%. There is a critical need for new therapies that increase efficacy without increasing toxicity. One therapeutic target can be found in the aberrant metabolism that is a hallmark of cancer. We have previously used an intracranial syngeneic immunocompetent mouse model of malignant glioma to demonstrate that targeting metabolism using a ketogenic diet reduced tumor burden and increased survival. The ketogenic diet is a high fat, low carbohydrate plus protein diet used for the treatment of refractory pediatric epilepsy. Mice maintained on a ketogenic diet (KetoCal®, Nutricia North America, Gaithersburg, MD) had a reduction in inflammation and peri-tumoral edema, prompting us to question the effects of the ketogenic diet on the infiltration and the functionality of glioblastoma-reactive immune cells. Immune cells were isolated from malignant gliomas from mice treated with either the ketogenic diet or a standard diet. Glioblastoma-specific immune cells were tested for functionality via cytokine production, capacity for cytotoxicity, and amount of infiltration. We found the ketogenic diet significantly decreased expression of the inhibitory receptors CTLA4 and PD-1 on tumor-infiltrating T cells, and coincided with a reduction in inhibitory ligand expression on the tumor. Moreover, this reduced T cell tolerance at the tumor site allowed CD8+ T cells to produce IFNγ and IL-2, and regain cytotoxic functions. Additionally, the ketogenic diet caused a reduction in immunosuppressive cytokine production via regulatory T cells. Innate immune responses to the tumor were also altered in mice maintained on the ketogenic diet, as natural killer cells infiltrating the tumor were able to produce more IFNγ and TNF in response to GL261-luc2 cells in vitro. Overall, the ketogenic diet may be an attractive adjuvant therapy to overcome several immune escape mechanisms in gliomas by decreasing regulatory T cell suppression of effector T cells and increasing CD8+ T cell killing and cytokine production, ultimately leading to increased tumor immune mediated rejection. Citation Format: John L. Johnson, Eric Woolf, Danielle M. Lussier, Ken S. Brooks, Joseph N. Blattman, Adrienne C. Scheck. Ketogenic diet enhances immunity to glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B73.

Chemotherapy of WAP-T mouse mammary carcinomas aggravates tumor phenotype and enhances tumor cell dissemination.

In this study, the effects of the standard chemotherapy, cyclophosphamide/adriamycin/5-fluorouracil (CAF) on tumor growth, dissemination and recurrence after orthotopic implantation of murine G-2 cells were analyzed in the syngeneic immunocompetent whey acidic protein-T mouse model (Wegwitz et al., PLoS One 2010; 5:e12103; Schulze-Garg et al., Oncogene 2000; 19:1028-37). Single-dose CAF treatment reduced tumor size significantly, but was not able to eradicate all tumor cells, as recurrent tumor growth was observed 4 weeks after CAF treatment. Nine days after CAF treatment, residual tumors showed features of regressive alterations and were composed of mesenchymal-like tumor cells, infiltrating immune cells and some tumor-associated fibroblasts with an intense deposition of collagen. Recurrent tumors were characterized by coagulative necrosis and less tumor cell differentiation compared with untreated tumors, suggesting a more aggressive tumor phenotype. In support, tumor cell dissemination was strongly enhanced in mice that had developed recurrent tumors in comparison with untreated controls, although only few disseminated tumor cells could be detected in various organs 9 days after CAF application. In vitro experiments revealed that CAF treatment of G-2 cells eliminates the vast majority of epithelial tumor cells, whereas tumor cells with a mesenchymal phenotype survive. These results together with the in vivo findings suggest that tumor cells that underwent epithelial-mesenchymal transition and/or exhibit stem-cell-like properties are difficult to eliminate using one round of CAF chemotherapy. The model system described here provides a valuable tool for the characterization of the effects of chemotherapeutic regimens on recurrent tumor growth and on tumor cell dissemination, thereby enabling the development and preclinical evaluation of novel therapeutic strategies to target mammary carcinomas.

ME-15 * INTERACTION BETWEEN CANCER STEM CELLS AND MYELOID DERIVED SUPPRESSOR CELLS DRIVES IMMUNOSUPPRESSION AND GLIOBLASTOMA GROWTH

Cellular and molecular regulation of the immune system is exquisitely controlled in the brain and disrupted in neoplasia. Glioblastoma (GBM) has an immunosuppressive phenotype, and despite the accumulation of immune cells in the tumor microenvironment, tumor growth persists. Myeloid derived suppressor cells (MDSCs) are a class of immature immune cells that mitigate cytotoxic T cell responses and promote immunosuppression in a variety of cancers. MDSCs are increased in the peripheral blood of GBM patients, but their role in the GBM microenvironment is undefined. We observed co-localization between arginase (Arg1) producing immunosuppressive MDSCs and cancer stem cells (CSCs) in GBM patient specimens and xenografts, leading to the hypothesis that CSCs stimulate MDSCs to promote immunosuppression. Intracranial injections of CSCs into mice led to GBM formation, expansion of the Arg1-producing MDSCs within the marrow of GBM-bearing mice, and increased levels of MDSCs within the GBM microenvironment. CSC conditioned media increased Arg1 production of murine marrow MDSCs which were then able to modulate cytotoxic T cell responses in co-culture models. Screening of conditioned media revealed cytokines secreted selectively by CSCs that activated MDSCs suppression. Removal of CSC-secreted factors decreased MDSCs in the GBM microenvironment, increased cytotoxic T cell infiltration, and had a pronounced survival benefit. Peripheral targeting of MDSCs through a sub-therapeutic dose of 5-fluorouracil (5-FU) in an immunocompetent syngeneic mouse model led to a similar phenotype to CSC-secreted factor removal in the GBM microenvironment, and an even greater survival benefit. Taken together, our results suggest a critical role of MDSC-mediated immunosuppression in GBM, a novel paradigm of immunomodulation by CSCs, and demonstrate that peripheral removal of MDSCs leads to increased T cell infiltration into GBM, and consequently suggest a novel chemotherapeutic strategy that is not generally toxic to bone marrow and that is not dependent on agents crossing the blood brain barrier.

Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade.

BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8(+) T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten(-/-) syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8(+) T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8(+) T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy.

Highlights of This Issue

Antibody-mediated pharmacodelivery of potent payloads represents a rapidly growing strategy in the field of cancer therapeutics. In this study, Gutbrodt and colleagues report the combination of antibody–drug conjugates (ADC) and interleukin-2–based immunocytokines targeting the alternatively-spliced extradomain A (EDA) of fibronectin. The combination promoted complete tumor eradications and conferred protective immunity in a syngeneic immunocompetent mouse model of leukemia, demonstrating, for the first time, that ADCs and immunocytokines can synergize. Since the EDA of fibronectin is commonly expressed in the majority of human solid tumors, lymphomas, and acute leukemias, the combination of these armed antibodies may have potential therapeutic applications in cancer.Metronomic chemotherapy has recently been recognized to have better therapeutic ratio compared to conventional therapeutic approaches. However, its efficacy in combination with an antiangiogenic treatment has not been well studied. Here, Gharpure and colleagues used metronomic docetaxel incorporated in PLGA-PRINT nanoparticles in combination with EZH2 siRNA (murine sequence) incorporated in chitosan nanoparticles. In ovarian cancer mouse models, the combination therapy effectively reduced tumor burden via reduced angiogenesis and proliferation and increased apoptosis. These results suggest that this combination therapy holds great potential for ovarian cancer therapy.Intrinsic or compensatory HER3 signaling may contribute to resistance to the EGFR monoclonal antibody cetuximab in head and neck squamous cell carcinoma (HNSCC). Here, Jiang and colleagues investigated the therapeutic benefit of combining MM-121/SAR256212, an anti-HER3 monoclonal antibody, with cetuximab in HNSCC models. The results revealed that the combination of cetuximab and MM-121 enhanced antitumor activity both in vitro and in vivo through simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways. This study provides a rationale for clinical application of HER3 inhibitor with EGFR-targeted therapies in HNSCC.Therapeutic targeting of mutant BRAF suppresses pro-growth signaling of the MAPK pathway. Parker and colleagues used a mass spectrometry screen of phosphoproteins to uncover previously unknown responses of BRAF inhibition. Bioinformatics was used to predict kinase families most likely responsible for regulating these changes. The authors showed that the upregulation of protein kinase CK2 family substrates was a consequence of blocking mutant BRAF, and further demonstrated antiproliferative synergy through the combinational blockade of BRAF and CK2 in BRAF-mutant melanomas and thyroid cancer cells. This highlights the potential of dual CK2/BRAF inhibition as a novel therapeutic strategy for cancers with BRAF mutation.