Syngeneic Rats Research Articles

Radiation therapy combined with intracerebral convection-enhanced delivery of cisplatin or carboplatin for treatment of the F98 rat glioma.

The purpose of this review is to summarize our own experimental studies carried out over a 13-year period of time using the F98 rat glioma as model for high grade gliomas. We evaluated a binary chemo-radiotherapeutic modality that combines either cisplatin (CDDP) or carboplatin, administered intracerebrally (i.c.) by means of convection-enhanced delivery (CED) or osmotic pumps, in combination with either synchrotron or conventional X-irradiation. F98 glioma cells were implanted stereotactically into the brains of syngeneic Fischer rats. Approximately 14days later, either CDDP or carboplatin was administered i.c. by CED, followed 24h later by radiotherapy using either a synchrotron or, subsequently, megavoltage linear accelerators (LINAC). CDDP was administered at a dose of 3µg in 5 µL, followed 24h later with an irradiation dose of 15Gy or carboplatin at a dose of 20µg in 10 µL, followed 24h later with 3 fractions of 8Gy each, at the source at the European Synchrotron Radiation Facility (ESRF). This resulted in a median survival time (MeST) > 180days with 33% long term survivors (LTS) for CDDP and a MeST > 60days with 8 to 22% LTS, for carboplatin. Subsequently it became apparent that comparable survival data could be obtained with megavoltage X-irradiation using a LINAC source. The best survival data were obtained with a dose of 72µg of carboplatin administered by means of Alzet® osmotic pumps over 7days. This resulted in a MeST of > 180days, with 55% LTS. Histopathologic examination of all the brains of the surviving rats revealed no residual tumor cells or evidence of significant radiation related effects. The results obtained using this combination therapy has, to the best of our knowledge, yielded the most promising survival data ever reported using the F98 glioma model.

Abstract 6136: Generation and characterization of colorectal cancer cell lines from the Buffalo rat

Abstract Introduction: Rat models of metastatic colorectal cancer (mCRC) to the liver are ideal for the development of transarterial therapies. To date, only one model of CRC cells grown in vitro and implanted into the liver of syngeneic rats is available. Yet, the small size of the WAG-Rij rat limits the use of the CC-531/WAG-Rij model for transarterial interventions. This work was aimed at generating an analogous model on the Buffalo rat, strain that has successfully been used for transarterial interventions in primary liver cancer. Materials and Methods: To induce CRC, 5 6-week old Buffalo rats were treated with Azoxymethane (AOM) and Dextran Sodium Sulfate (DSS). To assess tumor progression, the rats were regularly examined by colonoscopy as well as to T1- and T2-MR imaging from w20 up to w63. Standard procedures of tumor cell isolation and in vitro expansion were used. Once the polyclonal lines were established, expression of E-cadherin, Ceacam5, Mucin1, Vimentin1, and S100a4 was evaluated by flow cytometry (FACS) or qPCR. Additionally, the gene regions of APC (nt. 3049 to 3697) and β-catenin (nt. 371 to 1104) were analyzed by Sanger sequencing. Lastly, 23 adult Buffalo rats received implantations of 0.4-1 × 107 cells in the liver only or in the liver as well as subcutaneously. Results: Colonoscopies, MRIs, and histopathology demonstrated that all 5 rats treated with AOM/DSS developed colorectal neoplasms by w22. Tumors were harvested at various time points, at weeks 24, 32, 45, and 63. 2 polyclonal cell lines derived from invasive adenocarcinomas of a female (18L002) and a male (18L004) were successfully established and have proliferated in vitro >1year. FACS and qPCR results showed that both lines express the epithelial markers E-cadherin, Ceacam5, and Mucin1. Interestingly, despite high E-cadherin expression in both lines (>90% by FACS), qPCR data revealed that both lines also express the mesenchymal markers Vimentin and S100a4, suggestive of a trans-differentiated phenotype. DNA sequences of regions of the APC and β-catenin genes frequently mutated by AOM treatment appeared to be intact in both lines. MRIs of the 23 rats that received implants showed that 4 seem to have allowed for short-term growth (~14d) of the 18L004 cells in the liver before being re-absorbed. Histopathological analyses showed evidence of a serosal reaction at the site of implantation in those cases. Conclusions: Two polyclonal CRC lines with phenotypical characteristics of trans-differentiated cells were successfully generated from AOM/DSS-treated Buffalo rats. In spite of continued in vitro growth for over a year, neither line was tumorigenic in vivo, potentially due to the lack of mutations in key regions of the APC and β-catenin genes. Forced expression of mutant genes known to drive CRC progression such as β-catenin, Kras, or P53 could potentially increase in vivo tumorigenicity and allow for the establishment of a rat model of mCRC suitable for the study of transarterial therapies. Citation Format: Nina M. Muñoz, Crystal J. Dupuis, Katherine A. Dixon, Malea L. Williams, Kiersten L. Maldonado, Asif Rashid, Alda L. Tam. Generation and characterization of colorectal cancer cell lines from the Buffalo rat [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6136.

Attenuating ischemia/reperfusion injury in rat cardiac transplantation by intracoronary infusion with siRNA cocktail solution.

Tumor necrosis factor-α (TNF-α), caspase-8, and complement component 5a receptor (C5aR) are known to play a crucial role in the myocardial ischemia/reperfusion (I/R) injury in cardiac transplantation. We hypothesized that the intracoronary infusion of TNF-α, caspase-8, and C5aR small interfering RNAs (siRNA) would protect cardiac allograft function and improve graft survival from I/R injury-induced organ failure. I/R injury of cardiac allograft was induced by syngeneic rat cardiac transplantation, in which the transplanted hearts were infused with saline or different amounts of siRNA cocktail solution targeting TNF-α, caspase-8, and C5aR via coronary arteries, and subsequently subjected to 18 h of preservation at 4°C in histidine–tryptophan–ketoglutarate (HTK) solution. The effects of siRNA cocktail solution on prolonged cold I/R injury were determined by assessing graft survival, histopathological changes, myeloperoxidase (MPO) activity, and malondialdehyde (MDA) concentration. The perfused siRNA cocktail solution successfully knocked down the expression of TNF-α, caspase-8, and C5aR in vitro and in vivo. Approximately 91.7% of control hearts that underwent 18 h of cold ischemia ceased their function after transplantation; however, 87.5% of cardiac allografts from the highest dose siRNA cocktail solution-pretreated hearts survived >14 days and exhibited minimal histological changes, with minimal cellular infiltration, interstitial edema, and inflammation and maximal reduced MPO activity and MDA concentration in the cardiac allograft. We demonstrated the feasibility and efficiency of infusion of TNF-α, caspase-8, and C5aR siRNA via the intracoronary route as a promising strategy for gene silencing against I/R injury in cardiac transplantation.

Abstract 226: Recellularization of Xenograft Heart Valves Reduces the Xenoreactive Immune Response in an In Vivo Rat Model

Background: Current xenograft valve constructs provoke an intense immune response that may lead to valve dysfunction. Our aim was to address the role of autologous mesenchymal stem cell (MSC) recellularization of xenogenic valves on the activation of the xenoreactive immune response in an in-vivo rat model. Methods: Explanted aortic valve constructs from female Hartley guinea pigs were procured and decellularized, followed by recellularization with syngeneic Sprague-Dawley rat MSCs. The recellularized aortic valve xenografts were then implanted into the infrarenal aorta of recipient female Sprague-Dawley rats. Grafts were implanted as either syngeneic grafts, non-decellularized (fresh), decellularized and recellularized xenografts. Rats were euthanized after 7-days, exsanguinated and the grafts explanted. Total serum immunoglobulin was quantified and histological analysis perfomed to analyze the immune response. Results: Overall survival to endpoint was significantly lower in the decellularized xenograft group (67%; 4/6), compared to fresh (100%; 6/6) and recellularized grafts (100%; 6/6). Similarly grafts in the decellularized group were more likely to have completely thrombosed (50%; 2/4), compared to fresh (33%; 2/6) and recellularized grafts (0%; 0/6). Decellularized guinea pig xenografts, when implanted into rats in-vivo , result in significantly reduced total serum immunoglobulin production and significantly reduced graft cellular infiltrate when compared to fresh xenografts. Moreover, when decellularized guinea pig xenografts were recellularized with syngeneic rat MSCs there was an additional decrease in total serum immunoglobulin production and graft cellular infiltrate when compared to both fresh and decellularized xenografts. Importantly, recellularized guinea pig xenografts had an equivalent total immunoglobulin production and graft cellular infiltrate when compared to syngeneic rat aortic valve controls. Conclusions: Autologous MSC recellularization of xenogenic valves reduces the xenoreactive immune response in an in-vivo rat model and may be an effective approach to decrease the progression of xenograft valve dysfunction.

Liquid biopsy by NGS: Differential presence of exons (DPE) is related to metastatic potential in a colon-cancer model in the rat.

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.

The use of cryopreserved platelets in a trauma-induced hemorrhage model.

BackgroundCryopreserved platelet products can be stored for years and are mainly used in military settings. Following thawing, cryopreserved platelets are activated, resulting in faster clot formation but reduced aggregation in vitro, rendering their efficacy in bleeding unknown. Also, concerns remain on the safety of these products. The aim was to investigate the efficacy and safety of cryopreserved platelets in a rat model of traumatic hemorrhage.Study Design and MethodsAfter 1 hour of shock, rats (n = 13/group) were randomized to receive a balanced transfusion pack (1:1:1 red blood cell:plasma:platelet) made from syngeneic rat blood, containing either liquid stored platelets or cryopreserved platelets. Primary outcome was the transfusion volume required to obtain a mean arterial pressure (MAP) of 60 mmHg. Secondary outcomes were coagulation as assessed by thromboelastometry (ROTEM®) and organ failure as assessed by biochemistry and histopathology.ResultsThe transfusion volume to obtain a MAP of 60 mmHg was lower in animals receiving cryopreserved platelets (5.4 [4.1‐7.1] mL/kg) compared to those receiving liquid stored platelets (7.5 [6.4‐8.5] mL/kg, p < 0.05). ROTEM® clotting times were shorter (45 [41‐48] vs. 49 [45‐53]sec, p < 0.05), while maximum clot firmness was slightly lower (68 [67‐68] vs. 69 [69‐71]mm, p < 0.01). Organ failure was similar in both groups.ConclusionsUse of cryopreserved platelets required less transfusion volume to reach a targeted MAP compared to liquid stored platelets, while organ injury was similar. These results provide a rationale for clinical trials with cryopreserved platelets in (traumatic) bleeding.

Increased effect of two-fractionradiotherapy in conjunction with IDO1 inhibition in experimental glioblastoma.

ObjectivesThe aim of the study was to investigate therapeutic efficacy of single- or two-fraction radiotherapy in conjunction with IDO1-inhibition in a syngeneic rat glioblastoma model. IDO is known to cause immunosuppression through breakdown of tryptophan in the tumor microenvironment.MethodsGene expression analyses of IDO in glioblastoma were performed with data from publicly available datasets. Fractionation studies were done on animals to evaluate tumor size, immune cell infiltration of tumors and serum profile on day 18 after tumor inoculation. Survival analyses were done with animals carrying intracranial glioblastomas comparing two-fraction radiotherapy+IDO1-inhibition to controls. IDO inhibition was achieved by administration of 1-methyl tryptophan (1-MT), and radiotherapy (RT) was delivered in doses of 8Gy.ResultsThe expression of IDO1 was increased on gene level in glioblastoma stem cells. Tumor size was significantly reduced in animals treated with 1-MT+RTx 2 (both long and short intervals, i.e. 7 and 4 days between the treatments) as compared to control animals, animals treated with only 1-MT or animals treated with 1-MT+RTx1. Serum levels of IL-1A were significantly altered in all treated animals as compared to control animals. Survival was significantly increased in the animals treated with 1-MT+RTx2 (7-day interval) compared to control animals.ConclusionsAddition of two-fraction RT to IDO1 inhibition with 1-MT significantly reduced tumor size in animals with glioblastoma. Survival was significantly increased in animals treated with two-fractioned RT+1-MT as compared to untreated controls increased significantly.Advances in knowledgeThe currently used combination of only two fractions of radiotherapy and immune therapy is a promising area of research, increasing efficacy compared to single fraction irradiation, while potentially lowering radiation side effects compared to radiation in current clinical practice.

The BK activator NS11021 partially protects rat kidneys from cold storage and transplantation-induced mitochondrial and renal injury

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS induces mitochondrial and cellular injury, which exacerbates renal graft dysfunction, highlighting the need for therapeutic interventions. Using an in vitro model of renal CS, we recently reported that pharmacological activation of the mitochondrial BK channel (mitoBK) during CS protected against CS-induced mitochondrial injury and cell death. Here, we used an in vivo syngeneic rat model of renal CS (18 h) followed by transplantation (24 h reperfusion) (CS + Tx) to similarly evaluate whether addition of a mitoBK activator to the CS solution can alleviate CS + Tx-induced renal injury. Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from rat kidneys, and mitoBK protein level was reduced after CS + Tx compared to sham surgery. The addition of the BK activator NS11021 (3 μM) to the CS solution partially protected against CS + Tx-induced mitochondrial respiratory dysfunction, oxidative protein nitration, and cell death, but not acute renal dysfunction (SCr and BUN). In summary, the current preclinical study shows that pharmacologically targeting mitoBK channels during CS may be a promising therapeutic intervention to prevent CS + Tx-induced mitochondrial and renal injury.

A phase II study of selective HDAC6 inhibition with KA2507 for second-line treatment of advanced biliary tract cancer (ABC-11).

TPS4652 Background: The ABC-02 trial provided Level A evidence supporting the use of cisplatin plus gemcitabine as first-line chemotherapy for advanced biliary tract cancer (ABC) [Valle, 2010]. In second line therapy oxaliplatin and 5FU and ivosedinib for IDH1 mutated cancers are options [Lamarca, 2019; Abou-Alfa, 2019] however there remains significant unmet need for patients without actionable alterations. Histone deacetylase 6 (HDAC6) is over-expressed in cholangiocarcinoma, reducing primary cilia. This is mediated through increased resorption in normal human cholangiocytes via tubulin deacetylation in the ciliary axoneme. Inhibition of HDAC6 elicits both cell intrinsic and extrinsic anti-cancer activity. HDAC6 inhibition reversed oncogenic loss of ciliation and demonstrated preclinical efficacy in a syngeneic rat orthotopic biliary cancer model [Gradilone, 2013]. KA2507 is a potent and selective small molecule inhibitor of HDAC6. Phase I dose escalation study identified an oral dose of 800mg bid for further development, being well tolerated and showing evidence of selective target engagement. Methods: ABC-11 is a Phase II multi-centre, open-label study of KA2507 in 40 evaluable patients with advanced biliary tract cancer previously treated with standard of care chemotherapy. The study follows a single-arm single-stage design using A’Hern’s methodology. Eligible patients receive continuous 28-day cycles of fixed daily oral dose of KA2507 until death, disease progression or other pre-defined reason for study drug discontinuation. Tumour assessment is made at baseline and at 8-weekly intervals using RECIST 1.1 criteria until disease progression; primary endpoint is PFS at 4 months. Independent Data Monitoring Committee will review 4 month PFS and other data after first six patients, after a total of 17 patients (futility analysis, corresponding to cut-off of the Simon’s minimax 2-stage design; 33% was set as the target 4-month PFS rate expected with KA2507) and at least annually thereafter. Subject to availability of adequate tissue, mandatory pre-treatment and on-study tumour biopsy samples will undergo multiparameter flow cytometry of immune cell subsets, immunofluorescence analysis of immune cell subsets (activation status and topology) and T cell repertoire studies. The study received regulatory and ethical approval to proceed in January 2020 and enrolment is in progress. Clinical trial information: NCT04186156 .

A pre-investigational new drug study of lung spheroid cell therapy for treating pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a lethal interstitial lung disease with unknown etiology, no cure, and few treatment options. Herein, a therapy option is presented that makes use of a heterogeneous population of lung cells, including progenitor cells and supporting cells lines, cultured in adherent and suspension conditions, the latter of which induces spontaneous spheroid formation. Within these spheroids, progenitor marker expression is augmented. The cells, called lung spheroid cells, are isolated from fibrotic lungs, expanded, and delivered in single cell suspensions into rat models of pulmonary fibrosis via tail‐vein injections. Two bleomycin‐induced fibrotic rat models are used; a syngeneic Wistar‐Kyoto rat model, treated with syngeneic cells, and a xenogeneic nude rat model, treated with human cells. The first objective was to study the differences in fibrotic progression in the two rat models after bleomycin injury. Nude rat fibrosis formed quickly and extended for 30 days with no self‐resolution. Wistar‐Kyoto rat fibrosis was more gradual and began to decrease in severity between days 14 and 30. The second goal was to find the minimum effective dose of cells that demonstrated safe and effective therapeutic value. The resultant minimum effective therapeutic dose, acquired from the nude rat model, was 3 × 106 human cells. Histological analysis revealed no evidence of tumorigenicity, increased local immunological activity in the lungs, or an increase in liver enzyme production. These data demonstrate the safety and efficacy of lung spheroid cells in their application as therapeutic agents for pulmonary fibrosis, as well as their potential for clinical translation.

Glycocalyx‐Like Hydrogel Coatings for Small Diameter Vascular Grafts

AbstractNovel biological vascular conduits, such as decellularized tissue engineered vascular grafts (TEVGs) are hindered by high thrombogenicity. To mimic the antithrombogenic surface of native vessels with a continuous glycosaminoglycan layer that is present on endothelial cells (ECs), a hyaluronic acid (HA) modified surface is established, to effectively shield blood platelets from collagen‐triggered activation. Using the amine groups present on 4 mm diameter decellularized TEVGs, a continuous HA hydrogel coating is built via a bifunctional thiol‐reactive cross‐linker, thereby avoiding nonspecific collagen matrix cross‐linking. The HA hydrogel layer recreates a luminal wall, “hiding” exposed collagen from the bloodstream. In vitro blood tests show that adhered platelets, fibrinogen absorption, and fibrin formation on HA‐coated decellularized TEVGs are significantly lower than on uncoated decellularized TEVGs. The HA surface also inhibits macrophage adhesion in vitro. HA‐coated decellularized syngeneic rat aortae (≈1.5 mm diameter), and TEVGs in rat and canine models, respectively, are protected from aggressive thrombus formation, and preserve normal blood flow. Re‐endothelialization is also observed. HA‐coated TEVGs may be an off‐the‐shelf small‐diameter vascular graft with dual benefits: antithrombogenic protection and promotion of endothelium.

Abstract B87: Mechanistic insights into the antitumor activity of SB 11285—a novel STING agonist

Abstract Background: Cancer immunotherapy is a highly effective therapeutic option for cancer patients. However, the overall response rate with checkpoint inhibitors and other related modalities has been modest. Targeting innate immune signaling pathways that induce type I IFN production to reprogram tumor microenvironment and restore antitumor immunity represents a novel immunotherapeutic approach. We have previously reported that SB 11285 is a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous or intraperitoneal routes. Presented here are studies that provide additional insights into the mechanism of action of SB 11285 and analogs. Methods: (a) SB 11285 induces Type I IFN production and other cytokines in human PBMCs and PBMC-derived monocytes. PBMCs and monocytes, isolated from fresh PBMCs using pan monocyte isolation kit (Miltenyi Biotec), were stimulated with SB 11285. Type I IFNs and other cytokines were quantified using regular and multiplex ELISA assays. (b) SB 11285 directly binds STING. To address whether SB 11285 directly binds wild-type human STING, surface plasmon resonance assay was performed with a Biacore T200 device and a biotinylated SB 11285 analog (Biot-SB 11285). (c) Activity against STING polymorphs. Evaluation of SB 11285 and analogs was carried out using both SZ14 reporter cells (HEK293-derived) and HEK293T cells that stably or transiently express STING polymorphic variants. (d) Pharmacodynamic studies. Normal BALB/c mice were injected intravenously with SB 11285 or its analogs at 9 mg/kg. Serum, spleen, and liver samples were collected to quantify RANTES and TNF-α using ELISA. The expression of representative ISGs in spleen samples, including IRF7, IFIT2, and OAS1b were quantified using real-time PCR. (e) Determination of cellular uptake of SB 11285 by PBMCs. The STING agonist activity of Biot-SB 11285 in inducing type I IFN response was confirmed using SZ14 reporter cells and THP1-Dual-WT reporter cells. Human PBMCs were incubated with Biot-SB 11285 and the cellular uptake of compound by immune cells was evaluated using flow cytometry. Results and Conclusion: Our studies provide significant mechanistic insights into the STING agonistic activity of SB 11285 and analogs in that they: (a) induce Type I IFNs, other cytokines and chemokines in PBMCs and monocytes, monocyte-derived dendritic cells; (b) directly bind STING with nanomolar affinity; (c) activate multiple human STING polymorphic variants; (d) induce cytokines, chemokines, and ISGs in mice following i.v. injection; and (e) are effectively taken up by monocytes and other immune cells. SB 11285 is being advanced to human clinical trials. Citation Format: Shenghua Zhou, Sreerupa Challa, Vishal Nair, Geeta Meher, Anjaneyulu Sheri, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Leena Suppiah, Diane Schmidt, Santosh Khedkar, Radhakrishnan Iyer. Mechanistic insights into the antitumor activity of SB 11285—a novel STING agonist [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B87.

Abstract B96: Pharmacodynamic studies of SB 11285, a systemically bioavailable STING agonist in orthotopic tumor models

Abstract Background: The activation of innate and adaptive immunity via Stimulator of Interferon Genes (STING) signaling is a potentially transformative immunotherapeutic strategy in cancer. We have previously reported that SB 11285 is a novel and highly potent STING agonist that can be delivered by intravenous, intraperitoneal and intratumoral routes. We report here the antitumor and pharmacodynamic studies of SB 11285 in multiple orthotopic and subcutaneous syngeneic mouse tumor models. Methods: (1) Orthotopic NBT-II syngeneic rat bladder model: Rat bladder tumor cells were surgically implanted in the bladder wall of rats (n =10). After 6 days, SB 11285 was administered by i.v. @ 0.5, 1.5, and 3mg/kg on days 1, 5, 9,1 3, 17, 21, 25 and 31. Groups of rats treated with docetaxel at 7.5mg/kg i.v. and saline were used as positive and negative controls, respectively. At the end of dosing period, the bladders were harvested and evaluated by measurement of bladder weight and gross pathology. (2) Pharmacodynamic (PD) studies: Syngeneic mouse subcutaneous CT26 colon carcinoma and orthotopic 4T1 breast cancer models were used. In the CT26 model, when mean tumor volume (MTV) reached 125mm3, SB 11285 was administered at 3mg/kg i.v on days 1 and 5. In the orthotopic 4T1 model, SB 11285 was administered i.p. @ 10mg/kg on days 5, 7, 9, 11, 13, 17 and 19. Blood, lymph nodes, spleen and tumor were harvested and evaluated for (i) cytokine analysis by multiplexing assays and (ii) enumeration of CD4+, CD8+, Tregs, macrophages and MDSCs in blood, lymph nodes, spleen and tumor. Results: (1) In the orthotopic NBT-II rat bladder study, animals that received SB 11285 in all doses remained tumor-free based upon gross pathology and bladder weight measurements. SB 11285 was shown to be well tolerated and safe. (2) In both the 4T1 and CT26 PD studies, cytokine analysis of blood and tissues showed strong induction of type I interferon signature without significant systemic inflammatory response. Flow cytometric analysis of blood, lymph nodes, spleen and tumor revealed significant increase in macrophage &amp; CD8+ T-cell infiltration and decrease in Tregs &amp; CD4+ T-cell infiltration. Conclusion: SB 11285 showed potent antitumoral activity in multiple orthotopic and subcutaneous models that correlated with antitumoral immune response mediated by the induction of IFNs, and ISGs and other cytokines along with reduction of Tregs. SB 11285 is being advanced to human clinical trials. Citation Format: Sreerupa Challa, Balaji Ramachandran, Lalitha Vijayakrishnan, Douglas Weitzel, Shenghua Zhou, Kris Iyer. Pharmacodynamic studies of SB 11285, a systemically bioavailable STING agonist in orthotopic tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B96.

The Effect of Washing of Stored Red Blood Cell Transfusion Units on Post Transfusion Recovery and Outcome in a Pneumosepsis Animal Model.

Septic patients are often anemic, requiring red blood cell (RBC) transfusions. However, RBC transfusions are associated with organ injury. The mechanisms of RBC-induced organ injury are unknown, but increased clearance of donor RBCs from the circulation with trapping in the organs could play a role. We hypothesized that washing of RBCs prior to transfusion may reduce clearance and trapping of donor cells and thereby reduce organ injury. Sprague-Dawley rats were inoculated intratracheally with 10 colony-forming units (CFU) of Streptococcus pneumoniae or vehicle as a control and transfused with either a washed or standard (non-washed) biotinylated RBC transfusion from syngeneic rats. Controls received saline. Blood samples were taken directly after transfusion and at 24 h to calculate the 24 h post transfusion recovery (PTR). After sacrifice, flow cytometry was used to detect donor RBCs in organs and blood. The organs were histologically scored by a pathologist and CFUs in the lung and blood were counted. The 24h-PTR was similar between healthy and pneumoseptic rats after a standard transfusion. In healthy rats, a washed transfusion resulted in a higher PTR and less accumulation of donor RBCs in the organs compared with a standard transfusion. However, during pneumonia, this effect of washing was not seen. Transfusion did not further augment lung injury induced by pneumonia, but washing decreased bacterial outgrowth in the lungs associated with reduced lung injury. In healthy recipients, washing increased 24h-PTR of donor RBCs and decreased trapping in organs. In pneumoseptic rats, washing reduced bacterial outgrowth and lung injury, but did not improve PTR.

Comparing cellular bone matrices for posterolateral spinal fusion in a rat model.

IntroductionCellular bone matrices (CBM) are allograft products that provide three components essential to new bone formation: an osteoconductive scaffold, extracellular growth factors for cell proliferation and differentiation, and viable cells with osteogenic potential. This is an emerging technology being applied to augment spinal fusion procedures as an alternative to autografts.MethodsWe aim to compare the ability of six commercially‐available human CBMs (Trinity ELITE®, ViviGen®, Cellentra®, Osteocel® Pro, Bio4® and Map3®) to form a stable spinal fusion using an athymic rat model of posterolateral fusion. Iliac crest bone from syngeneic rats was used as a control to approximate the human gold standard. The allografts were implanted at L4‐5 according to vendor specifications in male athymic rats, with 15 rats in each group. MicroCT scans were performed at 48 hours and 6 weeks post‐implantation. The rats were euthanized 6 weeks after surgery and the lumbar spines were harvested for X‐ray, manual palpation and histology analysis by blinded reviewers.ResultsBy manual palpation, five of 15 rats of the syngeneic bone group were fused at 6 weeks. While Trinity ELITE had eight of 15 and Cellentra 11 of 15 rats with stable fusion, only 2 of 15 of ViviGen‐implanted spines were fused and zero of 15 of the Osteocel Pro, Bio4 and Map3 produced stable fusion. MicroCT analysis indicated that total bone volume increased from day 0 to week 6 for all groups except syngeneic bone group. Trinity ELITE (65%) and Cellentra (73%) had significantly greater bone volume increases over all other implants, which was consistent with the histological analysis.ConclusionTrinity ELITE and Cellentra were significantly better than other implants at forming new bone and achieving spinal fusion in this rat model at week 6. These results suggest that there may be large differences in the ability of different CBMs to elicit a successful fusion in the posterolateral spine.

Local and Systemic Antitumor Effects of Photo-activatable Paclitaxel Prodrug on Rat Breast Tumor Models.

We demonstrated that a large primary and a small untreated distant breast cancer could be controlled by local treatment with our light-activatable paclitaxel (PTX) prodrug. We hypothesized that the treated tumor would be damaged by the combinational effects of photodynamic therapy (PDT) and locally released PTX and that the distant tumor would be suppressed by systemic antitumor effects. Syngeneic rat breast cancer models (single- and two-tumor models) were established on Fischer 344 rats by subcutaneous injection of MAT B III cells. The rats were injected with PTX prodrug (dose: 1umolekg-1 , i.v.), and tumors were treated with illumination using a 690-nm laser (75 or 140mWcm-1 for 30min, cylindrical light diffuser, drug-light interval [DLI] 9h). Larger tumors (~16mm) were effectively ablated (100%) without recurrence for >90days. All cured rats rejected rechallenged tumor for up to 12months. In the two-tumor model, the treatment of the local large tumor (~16mm) also cured the untreated tumor (4-6mm) through adaptive immune activation. This is our first demonstration that local treatment with our PTX prodrug produces systemic antitumor effects. Further investigations are warranted to understand mechanisms and optimal conditions to achieve clinically translatable systemic antitumor effects.

Abstract B75: Nanoparticle formulation of the STING agonist SB 11285

Abstract Background: Immunotherapy has emerged as a transformative approach for the treatment of cancer. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in immune calls within the tumor microenvironment (TME) can induce the production of Interferons and cytokines that would lead to the induction of innate and adaptive immunity. We have previously disclosed the discovery and development of SB 11285 as a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous, or intraperitoneal routes. Presented here is the development of a controlled-release nanoparticle formulation of SB 11285 for subcutaneous administration. Methods: (a) Nanoparticle formulations of SB 11285. SB 11285-NPs were generated using Polylactic-glycolic acid copolymer (PLGA) using double emulsion and nano-precipitation methods (b) Imaging of nanoparticles: Scanning electron microscopy was performed to visualize and measure size of the nanoparticles. (c) Induction of IRF3 and NF-KB. THP-1 cells and RAW macrophages carrying dual reporter constructs were treated with either SB 11285-NPs or naked SB 11285 for 22hrs and induction of IRF3 and NF-KB was calculated as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs were treated with different concentrations of 11285-NPs or naked SB 11285, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA. (e) Dendritic cell maturation assay: PBMCs were differentiated into immature dendritic cells by addition of IL4 and GM-CSF followed by treatment with either SB 11285-NP or naked SB 11285. The maturation of dendritic cells was then evaluated by analyzing expression of CD83 and CD86 by flow cytometry. Results: PLGA formulations of SB 11285 prepared by double-emulsion method with an entrapment efficiency of 30 to 40% were spherical particles with an average diameter of 800nm. The nanoparticles were stable in aqueous media with minimal release of the entrapped SB 11285. In cell-culture studies, SB 11285-NPs showed potent induction of: (a) STING-dependent IRF3 induction in THP-1 cells (EC50: 3nM) as well as RAW macrophages (EC50: 3nM); (b) secretion of IFN-β (35 pg/ml), TNF-α (800 pg/ml), and RANTES (200 pg/ml), when tested at a low concentration of 112nM; and (c) SB 11285-NPs effectively induced DC maturation that was evident by increase in CD83 and CD86 expression. Conclusion: PLGA nanoparticles of SB 11285 were successfully prepared with submicron particle size that demonstrated potent expression of STING-dependent Type I IFN and cytokines in immune cells. The nanoparticles also induced maturation of dendritic cells. In vivo evaluation is in progress. Citation Format: Sreerupa Challa, Leena Suppiah, Dillon Cleary, Anjaneyulu Sheri, Rayomand Gimi, Geeta Meher, Seetharamaiyer Padmanabhan, Sumit Shah, Pranav Bhatt, Jovita Tauro, Radhakrishnan Iyer. Nanoparticle formulation of the STING agonist SB 11285 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B75.

Postnatal fate of donor mesenchymal stem cells after transamniotic stem cell therapy in a healthy model

PurposeWe sought to examine donor mesenchymal stem cell (MSC) fate after birth following transamniotic stem cell therapy (TRASCET) in a healthy model. MethodsLewis rat fetuses (n = 91) were divided into two groups based on the content of volume-matched intraamniotic injections performed on gestational day 17 (term = 21–22 days): either a suspension of amniotic fluid-derived MSCs (afMSCs) labeled with luciferase (n = 38) or acellular luciferase only (n = 53). Infused afMSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry. Samples from 14 anatomical sites (heart, lung, brain, liver, spleen, pancreas, bowel, kidney, thyroid, skin, skeletal muscle, thymus, peripheral blood and bone marrow) from survivors were screened for luciferase activity 16 days after birth. Statistical analysis was by logistic regression and the Wald test (p < 0.05). ResultsOverall survival was 32% (29/91). When controlled by the acellular luciferase injections, donor afMSCs were not identified at any anatomical site in any neonate as measured by relative light units (all p > 0.05). Donor afMSC viability was confirmed in term placentas. ConclusionsDonor mesenchymal stem cells are not detectable in the neonate after intraamniotic injection in a normal syngeneic rodent model. This finding suggests that clinical trials of transamniotic stem cell therapy may be amenable to regulatory approval. Level of evidenceN/A (animal and laboratory study).

Targeting of intragraft reactive oxygen species by APP-103, a novel polymer product, mitigates ischemia/reperfusion injury and promotes the survival of renal transplants.

Inflammatory responses associated with ischemia/reperfusion injury (IRI) play a central role in alloimmunity and transplant outcomes. A key event driving these inflammatory responses is the burst of reactive oxygen species (ROS), with hydrogen peroxide (H2 O2 ) as the most abundant form that occurs as a result of surgical implantation of the donor organ. Here, we used a syngeneic rat renal transplant and IRI model to evaluate the therapeutic properties of APP-103, a polyoxalate-based copolymer molecule containing vanillyl alcohol (VA) that exhibits high sensitivity and specificity toward the production of H2 O2 . We show that APP-103 is safe, and that it effectively promotes kidney function following IRI and survival of renal transplants. APP-103 reduces tissue injury and IRI-associated inflammatory responses in models of both warm ischemia (kidney clamping) and prolonged cold ischemia (syngeneic renal transplant). Mechanistically, we demonstrate that APP-103 exerts protective effects by specifically targeting the production of ROS. Our data introduce APP-103 as a novel, nontoxic, and site-activating therapeutic approach that effectively ameliorates the consequences of IRI in solid organ transplantation.

Heterotopic Implantation of Fetal Heart in Syngeneic Adult Rats (Comparative Study of Growth Conditions, Limits and Perspectives)

Heterotopic Implantation of Fetal Heart in Syngeneic Adult Rats (Comparative Study of Growth Conditions, Limits and Perspectives)