Syngeneic Grafts Research Articles

TNF-derived peptides inhibit tumour growth and metastasis through cytolytic effects on tumour lymphatics.

Tumour necrosis factor (TNF) is a multi-functional cytokine with profound and diverse effects on physiology and pathology. Identifying the molecular determinants underlying the functions and pathogenic effects of TNF is key to understanding its mechanisms of action and identifying new therapeutic opportunities based on this important molecule. Previously, we showed that some evolutionarily conserved peptides derived from TNF could induce cell death (e.g. apoptosis and/or necrosis), a feature of immune defence mechanisms shared by many vertebrates. In this study, we demonstrated that necrosis-inducing peptide P16 kills human glioblastoma cancer cells and primary human hepatoma or renal cancer cells isolated from patients who had not responded to standard treatments. Importantly, we show that the necrosis-inducing peptide P1516 significantly improves survival by inhibiting tumour metastasis in a 4T1 breast cancer syngeneic graft mouse model. Because the lymphatic system is an important metastatic route in many cancers, we also tested the effect of TNF-derived peptides on monolayers of primary human lymphatic endothelial cells (hDLEC) and found that they increased junctional permeability by inducing cytoskeletal reorganization, gap junction formation and cell death. Transmission electron microscopy imaging evidence, structural analysis and in-vitro liposome leakage experiments strongly suggest that this killing is due to the cytolytic nature of these peptides. P1516 provides another example of a pro-cytotoxic TNF peptide that probably functions as a cryptic necrotic factor released by TNF degradation. Its ability to inhibit tumour metastasis and improve survival may form the basis of a novel approach to cancer therapy.

Abstract 4618: A novel Pik3ca-driven mouse model and syngeneic cancer cell line for the preclinical testing of targeted and immune therapies for anal squamous cell carcinoma (ASCC)

Abstract Although many patients with localised anal squamous cell carcinoma (ASCC) initially achieve a complete response with standard chemoradiotherapy, those with persistent, relapsed or metastatic disease (35% of all patients) have limited treatment options and poor outcomes. Recent genomic profiling studies have identified PIK3CA as the most frequently mutated gene in anal cancers. Amplification of the PIK3CA gene and mutations in other PI3K pathway genes, have also been detected providing a strong rationale for targeting the PI3K pathway in these tumours. Similarly, the finding that many ASCC tumours express immune checkpoint receptors including PD-L1, has focussed attention on the potential use of checkpoint blockade in anal cancer. However, the use of targeted and/or immune therapies in the management of ASCC has been hampered by a lack of representative preclinical models for in vitro and in vivo testing of potential new therapeutic approaches. We have used mice with a Cre recombinase (Cre)-conditional knock-in of the Pik3caH1047R mutation and deletion of PTEN, crossed with mice expressing a tamoxifen-inducible Cre under the control of the ubiquitin C promoter, to generate a novel model of ASCC. By applying 4-hydroxy-tamoxifen topically to the anal canal we simultaneously induce expression of Pik3caH1047R and deletion of PTEN specifically in the anal epithelium. This results in anal tumors within 3 weeks with 100% penetrance. As all mice are on a C57Bl/6 background we are able to transplant the tumors subcutaneously into wild type C57Bl/6 mice as a syngeneic graft. To improve the utility of the model, we used tumors from these mice to establish a syngeneic mouse ASCC cell line. This line was then transduced with a human papilloma virus 16 E6/7 lentivirus to recapitulate the human virally-driven form of the disease. The cell line expresses both the Pik3ca mutation and E6/7 oncogenes and is positive for the squamous markers p63 and CK5/6. It had a similar response to 5-fluorouracil, Mitomycin C and radiotherapy, as a panel of human ASCC cell lines (also derived in our laboratory) and was sensitive to a PI3K inhibitor (BYL719). It was tumorigenic in both immunocompetent C57Bl/6 and immunodeficient NSG mice, additionally developing lung metastases in the NSG mice. The syngeneic C57Bl/6 tumors induce a peri-tumoral infiltrate, consisting of a heavy myeloid population, with high PDL1 expression, and a T-cell population expressing PD1; similar to human patients with treatment resistant disease. A major barrier to identifying new treatment options and improving overall survival in anal cancer has been the lack of preclinical models. We have now generated and characterised a novel, relevant Pik3ca-driven mouse model and syngeneic cancer cell line that will facilitate the in vitro and in vivo preclinical testing of targeted and immune therapies for ASCC. Citation Format: Glen R. Guerra, Sara Roth, Joseph C. Kong, Rosemary M. Millen, David S. Liu, Shienny Sampurno, Vignesh Narasimhan, Toan D. Pham, Karen G. Montgomery, Alexander G. Heriot, Robert G. Ramsay, Wayne A. Phillips. A novel Pik3ca-driven mouse model and syngeneic cancer cell line for the preclinical testing of targeted and immune therapies for anal squamous cell carcinoma (ASCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4618.

Abstract 5071: Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice

Abstract Palbociclib is the first CDK4/6-Cyclin D inhibitor approved by the FDA for ER+/Her2(erbB2)-breast cancer treatment. With the success of the drug in hormone-positive breast cancer, it is encouraging to explore more options of Palbociclib as an anticancer agent. erbB2 is a receptor tyrosine kinase (RTK) frequently overexpressed in breast cancer. The molecular hallmarks of erbB2+ breast cancer include the upregulation of cyclin D. The aim of this study was to explore the potential of Palbociclib as a preventive agent using MMTV-erbB2 transgenic mice, with a focus on the stemness of mammary epithelial cells (MECs) in the premalignant tissues. We first tested the in vivo effect of Palbociclib on tumor cells on MMTV-erbB2 mammary tumors using syngeneic tumor graft models. The animals bearing grafted tumors were treated with saline (control), low (75 mg/kg/day) and high (150 mg/kg/day) Palbociclib via oral gavage every 3 days for 29 days. We found that Palbociclib significantly inhibited tumor growth in a concentration dependent manner. To test the effect of Palbociclib on MEC stemness in premalignant mammary tissues, the animals at 9 wks of age were treated with saline, low and high (0, 75, 150 mg/kg/day) doses of Palbociclib every 3 days for 4 wks. We found the efficiency of both primary and secondary mammosphere formation of MECs from drug treated mice was significantly inhibited, suggesting the inhibition of mammary stemness in the premalignant tissues. Consistently, results from 3D cultures showed that colony numbers of the cells from drug treated mice were also lower than the control. Using CD24 and CD49f as markers, flow cytometry was performed to analyze the effect of Palbociclib on MEC subpopulations, including luminal, basal, and stromal populations. We showed that the percentage of luminal cells and putative mammary reconstitution units (MRUs) in drug treated mice was significantly inhibited. We also found that the percentage of CD61+/CD49+ cells, which are enriched with luminal progenitor cells and the origin of tumor initiation cells of this model, in Palbociclib treated tissues were also inhibited. The data suggest that Palbociclib induces MEC reprogramming in the premalignant tissues. Moreover, we found that the protein levels of pRb, E2F1, cyclin D1, c-myc, and cdc2 in cell cycle regulation, and pAKT, pERK, pmTOR, and p4EBP1 in the RTK pathway, pER, and DVL2 and LRP6 in the Wnt pathway were significantly downregulated in Palbociclib treated tissues, which was more evident in the high dose group. Taken together, our data indicate that Palbociclib inhibits MEC proliferation and stemness in the premalignant mammary tissues. Downregulation of cell cycle regulators and the signaling in ER, RTK, and Wnt pathway plays a critical role in this process. Our data support further investigation of Palbociclib in the prevention of ER+/ErbB2+ breast cancer. Citation Format: Amanda B. Parris, Yongxuan Liu, Zhikun Ma, Erin W. Howard, Xiaoshan Feng, Xiaohe Yang. Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5071.

Abstract 4991: Hyleukin-7, the Fc-fused interleukin-7, generates anti-tumor activity by modulating both adaptive and innate immune cells in the tumor microenvironment

Abstract Interleukine-7 (IL-7), a strong candidate for a novel immunotherapeutic agent, plays important roles in the development and homeostasis of T lymphocytes. Recombinant IL-7 has shown positive effects in various models by increasing T cells in both mice and humans; however, the short half-life and stability of recombinant IL-7 has remained a challenge for its clinical application to cancer immunotherapy. Here, we investigated anti-tumor effects of a long-acting form of recombinant human IL-7 fused with hybrid Fc (rhIL-7-hyFc; Hyleukin-7) in mice. rhIL-7-hyFc administration in tumor-free mice generated the cytokine-induced CD8+ T cell proliferation, which altering CD8+ T cell homeostasis by expanding largely the TCM-phenotype CD8+ T cells displaying activation-induced attributes, such as Eomes, Granzyme B, CXCR3, and IFN-γ. When injected into mice with syngeneic tumor graft, rhIL-7-hyFc induced anti-tumor activity in a dose-dependent manner. rhIL-7-hyFc dramatically expands CD8+ T cells in the periphery and recruits effector CD8+ T cells in the tumor, yielding a high CD8+ T/Treg cell ratio in the tumor microenvironment (TME). rhIL-7-hyFc increases Ki-67 and granzyme-B expression but decreases expression levels of immune checkpoint molecules on CD8+ tumor-infiltrating lymphocytes (TILs). Surprisingly, rhIL-7-hyFc reduced myeloid-derived suppressor cells (MDSCs) in the TME, yielding the high CD8+ T/MDSC ratio. Collectively, rhIL-7-hyFc treatment confers anti-cancer activity by inducing a “CD8+ T cell infiltrated-inflamed-immune favorable” TME. The combination treatment of rhIL-7-hyFc with cyclophosphamide and immune checkpoint blockades showed enhanced anti-tumor efficacy in an advanced tumor model. Furthermore, we found that the anti-tumor activity of rhIL-7-hyFc was achieved under lymphopenic conditions by normalizing CD8+ T cell homeostasis. In sum, rhIL-7-hyFc generates an effective anti-tumor response through reconstructing CD8+ T lymphocytes; this activity was highly enhanced by combination therapies with the chemotherapeutics and immune checkpoint blockades. Our data suggests that rhIL-7-hyFc can be applied to various cancer immunotherapy regimens as a monotherapy or in combination partner with conventional and other immunotherapies. Citation Format: Ji-Hae Kim, Sung-Wook Hong, Young-Min Kim, Saet-byeul Jo, Man Kyu Ji, Yeon Kyung Oh, Han Wook Park, Sora Kim, Donghoon Choi, Byung Ha Lee, Se Hwan Yang, Young Chul Sung, Seung-Woo Lee. Hyleukin-7, the Fc-fused interleukin-7, generates anti-tumor activity by modulating both adaptive and innate immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4991.

Abstract 4772: Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer

Abstract Human Adenoviruses (hAd) have been broadly used as gene delivery tool in preclinical and clinical studies of cancer gene therapy. The main challenges associated with systemic delivery of hAds are their high immunogenicity and host-specificity. Adenoviruses can elicit strong innate and acquired immune responses that reduce therapeutic gene transfer and expression in malignant sites. Murine cells generally lack some of the receptors necessary for hAd adhesion and internalization making them not permissive to infection and replication and limiting translational studies using murine models. We have developed a gene transfer method, which uses lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to protect the hAds from the immune system and to deliver them to a targeted tissue, bypassing the necessity for specific receptors. The expression of the Ad-receptors (CAR, Integrins αvβ3 and αvβ5), transduction efficiency of Ad-GFP, and GFP transgene expression were measured in vitro in murine (TRAMP-C2) and human (DU145) prostate cancer cells lines by flow cytometry and immunofluorescence. The activation of the innate and acquired immune system following the injection of Ad-GFP/MBs complexes was evaluated in vivo in C57BL/6 mice healthy or bearing a TRAMP-C2 syngeneic tumor graft. ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6 and measure Neutralizing Antibodies. ELISpot assay was performed to measure the frequency of DBP-specific CD8+ T cells secreting INF-γ. We showed in vitro that murine TRAMP-C2 cells display an expression pattern of Ad-receptors comparable to human DU145 prostate cancer cells. We also demonstrated that both murine and human cells showed a dose dependent increase in the percentage of cells transduced by Ad-GFP at 24 hours. Additionally, we showed that TRAMP-C2 cells efficiently express the GFP transgene at 48 and 72 hours post transduction. To assess in vivo if our gene transfer method could effectively protect the Ads form both the innate and adaptive immunity, we injected C57BL/6 mice with the hAds-GFP/MBs complexes +/-US. Notably we did not observe activation of either innate (secretion of TNF- and IL-6 cytokines) or acquired immunity (neutralizing antibodies and presence of adenovirus-specific CD8+ T cells producing INF-γ). Our data provides evidence that the TRAMP-C2 syngeneic model of prostate cancer is a suitable system to study in immunocompetent mice the capability of the MBs/US to protect the Adenoviruses from the immune system while delivering them to a targeted site bypassing the requirement of specific receptors. Citation Format: Flavia De Carlo, Elliot T. Varney, Pier Paolo Claudio, Candace M. Howard. Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4772.

Mouse Model of Tracheal Replacement With Electrospun Nanofiber Scaffolds.

The clinical experience with tissue-engineered tracheal grafts (TETGs) has been fraught with graft stenosis and delayed epithelialization. A mouse model of orthotopic replacement that recapitulates the clinical findings would facilitate the study of the cellular and molecular mechanisms underlying graft stenosis. Electrospun nanofiber tracheal scaffolds were created using nonresorbable (polyethylene terephthalate + polyurethane) and co-electrospun resorbable (polylactide-co-caprolactone/polyglycolic acid) polymers (n = 10/group). Biomechanical testing was performed to compare load displacement of nanofiber scaffolds to native mouse tracheas. Mice underwent orthotopic tracheal replacement with syngeneic grafts (n = 5) and nonresorbable (n = 10) and resorbable (n = 10) scaffolds. Tissue at the anastomosis was evaluated using hematoxylin and eosin (H&E), K5+ basal cells were evaluated with the help of immunofluorescence testing, and cellular infiltration of the scaffold was quantified. Micro computed tomography was performed to assess graft patency and correlate radiographic and histologic findings with respiratory symptoms. Synthetic scaffolds were supraphysiologic in compression tests compared to native mouse trachea ( P < .0001). Nonresorbable scaffolds were stiffer than resorbable scaffolds ( P = .0004). Eighty percent of syngeneic recipients survived to the study endpoint of 60 days postoperatively. Mean survival with nonresorbable scaffolds was 11.40 ± 7.31 days and 6.70 ± 3.95 days with resorbable scaffolds ( P = .095). Stenosis manifested with tissue overgrowth in nonresorbable scaffolds and malacia in resorbable scaffolds. Quantification of scaffold cellular infiltration correlated with length of survival in resorbable scaffolds (R2 = 0.95, P = .0051). Micro computed tomography demonstrated the development of graft stenosis at the distal anastomosis on day 5 and progressed until euthanasia was performed on day 11. Graft stenosis seen in orthotopic tracheal replacement with synthetic tracheal scaffolds can be modeled in mice. The wide array of lineage tracing and transgenic mouse models available will permit future investigation of the cellular and molecular mechanisms underlying TETG stenosis.

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Cationized liposomal keto-mycolic acids isolated from Mycobacterium bovis bacillus Calmette-Guérin induce antitumor immunity in a syngeneic murine bladder cancer model.

Intravesical therapy using Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most established cancer immunotherapy for bladder cancer. However, its underlying mechanisms are unknown. Mycolic acid (MA), the most abundant lipid of the BCG cell wall, is suspected to be one of the essential active components of this immunogenicity. Here, we developed cationic liposomes incorporating three subclasses (α, keto, and methoxy) of MA purified separately from BCG, using the dendron-bearing lipid D22. The cationic liposomes using D22 were efficiently taken up by the murine bladder cancer cell line MB49 in vitro, but the non-cationic liposomes were not. Lip-kMA, a cationic liposome containing keto-MA, presented strong antitumor activity in two murine syngeneic graft models using the murine bladder cancer cell lines MB49 and MBT-2 in comparison to both Lip-aMA and Lip-mMA, which contained α-MA and methoxy-MA, respectively. Interestingly, Lip-kMA(D12), which was made of D12 instead of D22, did not exhibit antitumor activity in the murine syngeneic graft model using MB49 cells, although it was successfully taken up by MB49 cells in vitro. Histologically, compared to the number of infiltrating CD4 lymphocytes, the number of CD8 lymphocytes was higher in the tumors treated with Lip-kMA. Antitumor effects of Lip-kMA were not observed in nude mice, whereas weak but significant effects were observed in beige mice with natural killer activity deficiency. Thus, a cationized liposome containing keto-MA derived from BCG induced in vivo antitumor immunity. These findings will provide new insights into lipid immunogenicity and the underlying mechanisms of BCG immunotherapy.

Heterologous and cross-species tropism of cancer-derived extracellular vesicles.

Extracellular vesicles (EVs) are naturally occurring cargo delivery vesicles that have recently received considerable attention for their roles in intercellular communication in many physiological and pathological processes, including tumourigenesis. EVs generated by different tissues demonstrated specific homing: in particular, cancer-derived EVs showed a selective tropism for the tumor tissue from which the vesicles originated. For this property, EVs have been proposed as drug delivery tools for anti-cancer therapies, although the limited knowledge about their in vivo tropism hinders their therapeutic applications. The current study aimed to characterize the targeting properties of cancer-derived EVs in vitro and their biodistribution in vivo, by using an imaging approach.Methods: EVs were generated from: i) murine lung (LL/2) and colon (MC-38) cancer lines, ii) human lung cancer cell line (A549) and iii) human liver biopsy samples from healthy individuals. EVs were loaded with fluorescent dyes alone or in combination with a biopharmaceutical agent, the oncolytic adenovirus (OV), characterized for charge and size and tested for their activity in cancer cell lines. Finally, optical imaging was extensively applied to study in vivo and ex vivo the biodistribution of EVs originated from different sources in different mouse models of cancer, including xenograft, syngeneic graft and the MMTV-NeuT genetically modified animal.Results: We initially demonstrated that even loading EVs even with a large biopharmaceutical oncolytic viruses (OVs) did not significantly change their charge and dimension properties, while increasing their anti-neoplastic activity compared to the virus or EVs alone. Interestingly, this activity was observed even if the EVs derived from lung cancer were applied to colon carcinoma cell lines and vice versa, suggesting that the EV uptake occurred in vitro without any specificity for the cancer cells from which the vesicles originated. When administered i.v (intravenously) to the mouse models of cancer, the tumour-derived EVs, but not the EVs derived from a healthy tissue, demonstrated a selective accumulation of the fluorescence at the tumour site 24 h after injection; adding OVs to the formulation did not change the tumour-specific tropism of the EVs also in vivo. Most interestingly, the in vivo experiments confirmed the in vitro observation of the generalized tropism of tumour-derived EVs for any neoplastic tissue, independent of the tumour type or even the species originating the vesicles.Conclusions: Taken together, our in vitro and in vivo data demonstrate for the first time a heterologous, cross-species tumour-tropism for cancer-derived EVs. This finding challenges our current view on the homing properties of EVs and opens new avenues for the selective delivery of diagnostic/therapeutic agents to solid tumours.

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

Chronic lung allograft dysfunction (CLAD) is a fatal condition that limits survival after lung transplantation (LTx). The pathological hallmark of CLAD is obliterative bronchiolitis (OB). A subset of patients present with a more aggressive CLAD phenotype, called restrictive allograft syndrome (RAS), characterized by lung parenchymal fibrosis (PF). The mouse orthotopic single LTx model has proven relevant to the mechanistic study of allograft injury. The minor-alloantigen-mismatched strain combination using C57BL/10(B10) donors and C57BL/6(B6) recipients reportedly leads to OB. Recognizing that OB severity is a spectrum that may coexist with other pathologies, including PF, we aimed to characterize and quantify pathologic features of CLAD in this model. Left LTx was performed in the following combinations: B10→B6, B6→B10, B6→B6. Four weeks posttransplant, blinded pathologic semi-quantitative assessment showed that OB was present in 66% of B10→B6 and 30% of B6→B10 grafts. Most mice with OB also had PF with a pattern of pleuroparenchymal fibroelastosis, reminiscent of human RAS-related pathology. Grading of pathologic changes demonstrated variable severity of airway fibrosis, PF, acute rejection, vascular fibrosis, and epithelial changes, similar to those seen in human CLAD. These assessments can make the murine LTx model a more useful tool for further mechanistic studies of CLAD pathogenesis.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.

Survival and Metabolic Function of Syngeneic Mouse Islet Grafts Transplanted Into the Hepatic Sinus Tract.

Islet grafts are transplanted into the liver via a portal vein in 90% of the clinical islet transplantations. However, the portal vein is far from being the ideal infusion site due to its unique drawbacks. These issues necessitated the exploration of an alternatively optimized site for clinical islet transplantation. With the widespread clinical application of percutaneous transhepatic puncture technique, we envisioned the possibility of islet transplantation into the hepatic sinus tract (HST). The HST was created by temporarily placing a medically approved material into the hepatic parenchyma of C57BL/6 mice. The syngeneic islets were transplanted into the HST, after which, the nonfasting blood glucose, intraperitoneal glucose tolerance, and morphology were evaluated. A collagen-lined HST was formed by the 28-day implantation of a cylindrical nylon rod. Transplantation of ~300 syngeneic islets into the HST routinely reversed the hyperglycemia of the recipient mice and maintained normoglycemia for longer than 100 days until the graft was removed. The islet grafts within the HST stained positively for insulin, glucagon, and abundant microvessels and achieved comparable results to the islet grafts under the kidney capsule with respect to glycemic control and glucose tolerance. These results suggested that an HST can be constructed for islet transplantation by temporarily placing a nylon material in the liver parenchyma. The HST is a promising site for clinical islet transplantation, thereby providing a satisfactory environment for the survival and metabolic function of islet grafts.

Abstract PR03: Analysis of immune infiltrate identifies checkpoint blockade and TLR3 activation as efficient synergistic combination of immunotherapy in rhabdoid tumors

Abstract Rationale: Rhabdoid tumors (RTs) are highly aggressive cancers of infancy, arising from the central nervous system (atypical teratoid rhabdoid tumors, AT/RT) and other miscellaneous extracranial locations. RTs are characterized by the biallelic inactivation of the SMARCB1 tumor suppressor gene and an otherwise highly stable genome. Prospective trials based on intensive multimodal therapies have led to few survivals, and the side effects are a great matter of concern. In the prospect of new therapeutic strategies, we analyzed the immune contexture of both human and mouse RT. We subsequently explored in vivo targeting of the immune infiltrate in order to determine the best immunotherapy strategy adapted to the specific immune context. Methods: We first analyzed the immune infiltrate of a cohort of human tumor samples, including both AT/RT and extracranial RT, by immunohistochemistry, expression profiling, and flow cytometry. In parallel, we performed the same analyses on tumors obtained from the genetically engineered mouse model (GEMM) of RT Smarcb1flox/flox;Rosa26-CreERT2 previously established in our lab, and syngeneic heterotopic engraftment models that we derived from the GEMM. After identifying new therapeutic targets, we validated them in preclinical models of RT. Results: Despite a well-established low mutational burden and stable genome, we found that both human and mouse RT are highly infiltrated by immune cells of both lymphoid and myeloid lineages. Tumor-infiltrating lymphocytes consist in equal proportion of CD4+ and CD8+ T cells with few regulatory T cells. We observed that T cells coexpress PD-1 and TIM-3 activation/exhaustion markers, while tumor and myeloid cells express their respective ligands, i.e., PD-L1 and Galectin 9. Consistently, we found that blockade of the PD-1/PD-L1 pathway was able to impair tumor growth in syngeneic models of RT and induced memory against second engraftment. Furthermore, combination of anti-TIM-3 and anti-PD-1 shows synergistic activity. In the context of RT syngeneic engraftment, we observed the expansion of aberrant myelopoiesis corresponding to suppressive myeloid cells. Tumor-infiltrating myeloid transcriptomic signatures correlated with the expression of chemokines known to recruit such myeloid cells. We found that tumor-infiltrating myeloid cells were a particularly intense and constant feature both in human and mouse RT, consisting primarily of macrophages of a protumoral phenotype. Targeting this myeloid infiltrate by TLR3 activation with poly(I:C) induced a potent antitumor effect. Combination of poly(I:C) with PD-1 blockade had a synergistic effect, leading to complete tumor regression in around 90% of treated mice. Conclusion: We show here for the first time that rhabdoid tumors are highly immune-infiltrated tumors susceptible to PD-1/PD-L1 and TIM-3 blockade, and to reprogramming of myeloid suppressive cells. Immunogenicity of rhabdoid tumor cells is a surprising observation in the field of pediatric tumors; we hypothesize that rhabdoid tumor recognition by T cells is initiated by a SMARCB1-dependent mechanism under investigation. Citation Format: Amaury Leruste, Zhi-Yan Han, Arnault Tauziède-Espariat, Pamela Caudana, Joshua J. Waterfall, Mamy Andrianteranagna, Christine Sedlik, Rodrigo Ramos, Sophie Viel, Céline Chauvin, Olivier Delattre, Eliane Piaggio, Franck Bourdeaut. Analysis of immune infiltrate identifies checkpoint blockade and TLR3 activation as efficient synergistic combination of immunotherapy in rhabdoid tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR03.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 128. Comparative study of cellular bone matrices for posterolateral spinal fusion

BACKGROUND CONTEXT Cellular bone matrices (CBM) are allograft products that combine demineralized bone with viable cancellous bone prepared to retain cells with skeletal tissue differentiation capabilities. Using an athymic rat model of posterolateral fusion, several commercially-available human CBMs were compared: Trinity ELITE (TEL; OrthoFix, Lewisville, TX, United States), ViviGen (VIV; DePuy Synthes, Raynham, MA, United States), Cellentra (CEL; Zimmer Biomet, Warsaw, IN, United States), Osteocel Pro (OCP; NuVasive, San Diego, CA, United States), Bio4 (BIO; Stryker, Kalamazoo, MI, United States) and map3 (MAP; RTI Surgical, Marquette, MI, United States). Iliac crest bone from syngeneic rats was used as a control to approximate the human gold standard. METHODS To allow for lot-to-lot variability, three different lots of each implant type were procured and each was used to implant five rats, for a total of 15 9-10 week old male rats per implant type. Each implant was stored, thawed, and prepared according to the manufacturer's instructions and all implantations occurred within the manufacturers’ time allowance for use after preparation. After anesthesia and preparation, a single posterior midline longitudinal skin and subcutaneous incision and bilateral longitudinal paraspinal myofascial incisions were made to expose the transverse processes and intertransverse membranes at the L4-5 level. The processes were decorticated and 0.3 cc of allograft implant or freshly harvested syngeneic iliac bone graft was placed bilaterally. Surgeons were blinded as to which implant was which. Incisions were closed with sutures to avoid any interference with in vivo animal microCT scans performed within 48 hours of surgery on all rats. The rats were euthanized 6 weeks after surgery and the lumbar spines harvested. A second microCT scan was taken and compared with the microCT taken at the time of implantation to assess fusion and bone remodeling. Stable fusion was also evaluated by manual palpation by three independent, blinded reviewers. MicroCT analysis was performed by an independent CRO (ImageIQ, Cleveland, OH, USA). All assessments were done by experienced evaluators blinded to the treatments. Anonymity of implant type was rigorously kept to avoid any bias. RESULTS By manual palpation, 5/15 (33%) rats of the syngeneic bone group were fused at 6 weeks. While TEL had 8/15 (53%) and CEL 11/15 (73%) rats with stable fusion, only 2/15 (13%) of VIV-implanted spines were fused and 0/15 (0%) of the OCP, BIO and MAP CBMs produced stable fusion. The TEL and CEL groups were significantly different from the other CBMs but not different from each other. MicroCT analysis indicated percentage increases in bone volume from the first measurement to the second for all groups, but only TEL and CEL had significant increases over the syngeneic bone control (TEL 65% and CEL 73% vs. syngeneic bone 21%, respectively). CONCLUSIONS Because the CBMs tested have distinct formulations and are likely processed differently, it is not surprising that the results would be different between the groups. While map3 has cells added at the time of surgery, the other CBMs are processed with cells adherent to the bone matrix. The claimed live cell and stem cell contents differ between products. Trinity ELITE and Cellentra were found to be significantly better than other implants at forming new bone and achieving fusion. The other CBMs did not produce greater bone formation than the control and were very poor at forming a solid fusion. These results suggest that there may be large differences in the ability of different CBMs to elicit a successful fusion in the posterolateral spine. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

The atypical chemokine receptor-2 does not alter corneal graft survival but regulates early stage of corneal graft-induced lymphangiogenesis

PurposeTo re-evaluate the role of the atypical chemokine receptor-2 (ACKR2) in corneal graft rejection and investigate the effect of ACKR2 on inflammation-associated lymphangiogenesis using murine orthotopic corneal transplantation.MethodsCorneal grafts were performed and evaluated in the settings of syngeneic, allogeneic and single antigen (HY-antigen) disparity pairings. Corneal vessels were quantified in whole mounts from WT, ACKR2−/− and F4/80−/−ACKR2−/− mice that received syngeneic or allogeneic grafts using anti-CD31 and anti-Lyve-1 antibodies.ResultsSyngeneic corneal grafts in WT and ACKR2−/− mice were 100% accepted. Fully histo-incompatible allogeneic grafts were rapidly rejected (100%) with similar tempo in both WT and ACKR2−/− hosts. Around 50% of single-antigen (HY) disparity grafts rejected at a slow but similar tempo (60 days) in WT and ACKR2−/− mice. Prior to grafting, F4/80−/−ACKR2−/− mice had lower baseline levels of limbal blood and lymphatic vessels compared to ACKR2−/− mice. Syngeneic grafts, but not allogeneic grafts, in ACKR2−/− and F4/80−/−ACKR2−/− mice induced higher levels of lymphatic sprouting and infiltration of Lyve-1+ cells during the early (3d) post-graft (pg) stage but lymphatic density was similar to WT grafted mice by 7d pg.ConclusionsOur results indicate that the chemokine scavenger receptor, ACKR2, has no role to play in the survival of allogeneic grafts. A minor role in regulation of lymphangiogenesis in the early stage of wound healing in syngeneic grafts is suggested, but this effect is probably masked by the more pronounced lymphangiogenic inflammatory response in allogeneic grafts. No additional effect was observed with the deletion of the resident macrophage gene, F4/80.

PO-421 Anti-CTLA-4 and anti-PD-1 immunotherapies repress tumour progression through independent cellular mechanisms

IntroductionThe recent development of immunotherapy represents a significant breakthrough in cancer therapy. Several immunotherapies provide robust efficacy gains in a wide variety of cancers. However, in some patients immune checkpoint blockade remains ineffective due to absence of a therapeutic response, tumour relapse, and high severity of adverse drug reactions. An improved comprehension of the basic mechanisms underlying tumor-immune system interactions may improve clinical management of cancer. Here, we report a preclinical strategy developed in Porsolt to test two recent FDA-approved antibodies, e.g. anti-CTLA-4 and anti-PD-1. We demonstrate that the two antibodies can affect the tumour immune microenvironment differently even if they both display efficient anticancer activity.Material and methodsWe used in vivo mouse syngeneic grafting models of breast and colon cancer to evaluate tumour progression and metastasis formation. In addition we characterised post-mortem the tumour immune infiltration using high-throughput cytomety-based multiplex analysis to identify tumour microenvironment specific response to immunotherapies.Results and discussionsHere, we demonstrated that a treatment with anti-CTLA-4 or anti-PD-1 results in efficient control of tumour progression in a majority of mice. These antibodies also affect the metastasis niche. Finally, the immune microenvironment and in particular the tumor-infiltrating lymphocytes are differentially regulated by anti-CTLA-4 and anti-PD-1.ConclusionThis work reveals a key target-specific effect of CTLA-4 or PD-1 blockade on tumour progression and on tumor-infiltrating lymphocyte subpopulations. Our experimental approach provides a relevant preclinical strategy with a strong translational power to predict pharmacological response of immunotherapies as well as mechanisms of resistance and adverse effects that may affect patients in clinic.

Abstract 143: MAPK pathway blockade effects on glioma stem cells and immunotherapy in BRAFV600E mutant gliomas

Abstract BRAFV600E mutations are frequent in pediatric gliomas. We previously showed antitumor activity when using BRAF and/or MEK inhibitors against BRAFV600E glioma syngeneic grafts (Grossauer et al., Oncotarget 2016;7:775839). While early clinical trial reports suggest an encouraging response rate to BRAFV600E inhibitors alone (Burger et al., Oncol Rep 2017;38:3291) and in combination with MEK inhibition (Brown et al., CNS Oncol 2017:6:291), resistance has been reported in BRAFV600E mutant melanoma. Moreover, we reported that resistance to BRAFV600E inhibitor monotherapy occurs in mouse models and is driven by stem-like cells (Lerner et al., Cancer Res 2015;75:5355). Rapid reactivation of MEK pathway and prosurvival signaling via EGFR have been implicated as mechanisms for acquired resistance (Yao et al., Oncotarget 2017;8:583). It is just unclear why stem-like cells survive treatment better than non-stem like tumor cells and how they respond to additional therapies, such as MEK inhibition but also immune checkpoint inhibition. Immunocompetent, syngeneic FVB/N mice were intracranial-engrafted with luciferase-modified BRAFV600E mutant CDKN2A-deficient murine glioma cell line 2341luc and treated with BRAFV600E and/or MEK inhibitors, either alone or in combination, and either with or without PD-L1 (Clone 10F.9G2) and CTLA-4 (Clone 9D9) mAbs. While the combination of BRAF and MEK inhibitors significantly increased the median survival from 46 to 70 days (P &amp;lt;0.05), additional treatment with PD-L1 and CTLA-4 mAbs further increased survival to a median of 150 days (P &amp;lt;0.001). Notably, PD-1 and CTLA-4 blockade, alone, only marginally increased survival to 60.5 days (P &amp;lt;0.05). To understand the mechanism underlying the impact on survival, we examined the tumor microenvironment using CyToF as well as T cell and CD133+ cancer stem cell composition using flow cytometry in treated tumors. Our preliminary findings showed that the survival benefit of quadruple treatment associates with an increased frequency of CD8+ cells and a decrease in T regulatory cells and cancer stem cells. In summary, we report that in BRAFV600E mutant gliomas when MAPK pathway and immune checkpoint blockades are combined, an immune profile indicative of productive tumor immunity is observed. CyToF analyses of treated and untreated tumors and RNA sequencing analyses of stem and non-stem like tumor cells from untreated and BRAFV600E inhibitor treated mice are on the way. These data provide rationale for further understanding this therapeutic approach, as well as the potential for clinical consideration in the near future. Citation Format: Stefan Grossauer, Katharina Koeck, Malek Chouchane, Joanna J. Phillips, Claudia K. Petritsch, Theodore Nicolaides. MAPK pathway blockade effects on glioma stem cells and immunotherapy in BRAFV600E mutant gliomas [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 143.

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells

ObjectiveLactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the...

Syngeneic stem cell transplantation generates antigen-specific T cells that maintain myeloma-immune equilibrium

Abstract Multiple myeloma (MM) is a largely incurable hematological malignancy characterized by clonal expansion of plasma cells in bone marrow (BM). Autologous stem cell transplantation (ASCT) remains a standard treatment for MM, allows intensive cytoreduction and induces inflammation in the context of lymphodepletion. We hypothesized that SCT may overcome myeloma-induced immune defects to restore immune-equilibrium. To address this, we developed a model of ASCT for MM using Vk*MYC myeloma. Surprisingly, we demonstrate the induction of T cell-dependent MM control after SCT as recipients of syngeneic BM grafts containing T cells had prolonged survival and reduced tumor burden compared to BM alone. This anti-myeloma activity was independent of γδ T, natural killer (NK) and NK T cells and was dependent on CD4+and CD8+ T cells. MM-primed T cells, harvested from SCT recipients with controlled MM, protected against relapse in secondary recipients and were Vk*MYC clone-specific. Recipients of MM-primed T cells had increased central memory CD8+ T cells and transfer of MM-primed CD8+ T cells was sufficient to prevent relapse. Furthermore, TCR sequencing of CD8+ T cells from mice with controlled MM revealed a higher clonotype overlap relative to MM-free SCT recipients, consistent with MM-specific T cells. Donor IFNγ secretion and signaling was critical to protective immunity and CD8+ T cells from mice with relapsed MM had impaired IFNγ production compared to those with controlled disease. Importantly, IFNγ production was profoundly augmented by CD137 agonist treatment such that majority of mice controlled disease. These data provide new insights into the mechanisms of action of SCT in myeloma and suggest the use of CD137 agonists to prevent relapse.