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APS001F is a strain of Bifidobacterium longum genetically engineered to express cytosine deaminase that converts 5-fluorocytosine (5-FC) to 5-fluorouracil. In the present study, antitumor effects of APS001F plus 5-FC (APS001F/5-FC) in combination with anti-PD-1 monoclonal antibody were investigated using a CT26 syngeneic mouse model. Both of dosing of APS001F/5-FC before and after anti-PD-1 mAb in the combination dosing exhibited antitumor effects as well as prolonged survival over the nontreated control. The survival rate in the combination therapy significantly increased over the monotherapy with APS001F/5-FC and that with anti-PD-1 mAb. Regulatory T cells among CD4+ T cells in tumor decreased in the combination therapy, while the ratio of CD8+ T cells was maintained in all groups. Taken these results together, APS001F/5-FC not only demonstrates a direct antitumor activity, but also immunomodulatory effects once localized in the hypoxic region of the tumor, which allows anti-PD-1 mAb to exert potentiated antitumor effects.

BackgroundInterferon α (IFNα) was the first cytokine clinically tested as a cancer therapy. IFNα is a member of the type-I IFN family and activates immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. High dose IFNα therapy was approved for melanoma, lymphoma, and leukemia but its use is limited by systemic toxicity and modest efficacy.MethodsWTX-613 is a novel systemically delivered IFNα2b pro-drug identified using the Predator™ discovery platform. The inducible WTX-613 INDUKINE™ molecule is designed to deliver wild-type IFNα2b in the tumor microenvironment to reduce systemic toxicity. WTX-613 has two identical half-life extension (HLE) domains tethered to IFNα2b via a tumor protease-sensitive linker. The HLE domain supports less frequent systemic administration but importantly also prevents binding of WTX-613 to IFNAR due to steric hindrance until removal of the HLE domains by tumor proteases.ResultsWTX-613 was selected as a lead molecule due to its improved in vitro profile. Since human IFNα is not functional in the mouse, a surrogate WTX-613 molecule was created consisting of mouse IFNα1 to explore anti-tumor responses in mouse syngeneic tumor models. Intraperitoneal (i.p.) administration of the WTX-613 surrogate resulted in anti-tumor responses in the more immunogenic MC38 colon model which was well tolerated by the mice. Furthermore, less immunogenic tumor models such as B16F10 melanoma and EMT6 breast carcinoma, which are generally less responsive to I/O therapy, also responded with similar anti-tumor activity. Importantly, wild-type mouse IFNα1 was only active in mouse models during the dosing period, and tumors grew back once treatment was stopped. However, the WTX-613 surrogate INDUKINE™ molecule had long lasting anti-tumor activity when dosed at equimolar amounts compared to the native IFNα during the dosing period. The WTX-613 surrogate strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in MC38 tumors. Specifically, the WTX-613 surrogate was better than native IFNα1 in inducing CD8+, NK, and DC cells.ConclusionsPreclinical data obtained so far support the continued development of this innovative and differentiated engineered IFNα therapy and progression into clinical trials.Ethics ApprovalAll mouse in vivo work was performed in accordance with current regulations and standards of the U.S. Department of Agriculture and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA).

Despite the recognition of the lethality of cancer metastasis and the importance of developing specific anti-metastasis therapies directed at the cancer metastatic cascade, the dynamics of cancer metastasis remains poorly understood. In this study, we examined the dynamics of circulating tumor cell (CTC) survival in the bloodstream using experimental mouse models. CTCs were arrested in the capillaries by adhesion to vascular endothelium within a few minutes after injection into the bloodstream. The loss of CTCs from the circulation followed a bi-phasic decay pattern, with the number of CTCs in the bloodstream being closely associated with the number of blood circulation cycles. The calculated in vivo Vd (apparent volume of distribution) of the CTC revealed organ specific binding of the CTCs. Moreover, confocal microscopy, in vivo fluorescence imaging in syngeneic mouse metastatic models and analysis of blood circulation patterns support the notion of organ-specific tumor metastasis. The present study suggests that organ-specific tumor metastasis is influenced by cooperation between blood circulation patterns and 'seed-soil' compatibility factors. These new findings provide further insights for optimized cancer metastatic prevention strategies such as by creating a hostile circulation microenvironment and targeting the organ-specific 'seed-soil' compatibility factors.

Nutrition can modulate host immune responses as well as promote anticancer effects. In this study, two nutritional supplements, namely gamma-tocotrienol (γT3) and Spirulina, were evaluated for their immune-enhancing and anticancer effects in a syngeneic mouse model of breast cancer (BC). Five-week-old female BALB/c mice were fed Spirulina, γT3, or a combination of Spirulina and γT3 (Spirulina + γT3) for 56 days. The mice were inoculated with 4T1 cells into their mammary fat pad on day 28 to induce BC. The animals were culled on day 56 for various analyses. A significant reduction (p &lt, 0.05) in tumor volume was only observed on day 37 and 49 in animals fed with the combination of γT3 + Spirulina. There was a marked increase (p &lt, 0.05) of CD4/CD127+ T-cells and decrease (p &lt, 0.05) of T-regulatory cells in peripheral blood from mice fed with either γT3 or Spirulina. The breast tissue of the combined group showed abundant areas of necrosis, but did not prevent metastasis to the liver. Although there was a significant increase (p &lt, 0.05) of MIG-6 and Cadherin 13 expression in tumors from γT3-fed animals, there were no significant (p &gt, 0.05) differences in the expression of MIG-6, Cadherin 13, BIRC5, and Serpine1 upon combined feeding. This showed that combined γT3 + Spirulina treatment did not show any synergistic anticancer effects in this study model.

Pancreatic ductal adenocarcinoma (PDAC) is in urgent need of better diagnostic and therapeutic methods due to its late diagnosis, limited treatment options and poor prognosis. Finding the right animal models to recapitulate the tumor molecular pathogenesis and tumor microenvironment (TME) complexity is critical for preclinical immunotherapeutic and non-immunotherapeutic treatment developments. In this review, we summarize and evaluate popular preclinical animal models including patient-derived xenograft models, humanized mouse models, genetically engineered mouse models, and syngeneic mouse models. Through comparisons between these models in different research settings, we hope to provide guidance in finding the most relevant preclinical models to suit various research purposes.

Anti-viral immunity presents a major hurdle for systemically administered oncolytic viruses (OV). Intratumoral OV therapy has a potential to overcome this problem through activation of anti-tumor immune response, with local and abscopal effects. However, the effects of anti-viral immunity in such a setting are still not well defined. Using Newcastle Disease Virus (NDV) as a model, we explore the effects of pre-existing anti-viral immunity on therapeutic efficacy in syngeneic mouse tumor models. Unexpectedly, we find that while pre-existing immunity to NDV limits its replication in tumors, tumor clearance, abscopal anti-tumor immune effects, and survival are not compromised and, on the contrary, are superior in NDV-immunized mice. These findings demonstrate that pre-existing immunity to NDV may increase its therapeutic efficacy through potentiation of systemic anti-tumor immunity, which provides clinical rationale for repeated therapeutic dosing and prompts investigation of such effects with other OVs.

Introduction: Chimeric antigen receptor-based T cell therapy (CAR-T) has shown great promise in B cell malignancies. CD30-targeted therapies like brentuximab vedotin have activity in T cell malignancies expressing CD30. There is interest in developing CD30-targeted CAR-T therapies for the treatment of relapsed T cell malignancies. As the process of generating autologous T cells takes several weeks, there is a need for active bridging therapeutic regimens that can reduce tumor burden to facilitate subsequent treatment with CAR-T cell therapy. We propose the use of devimistat, a TCA cycle inhibitor, in combination with bendamustine as bridging therapy for the treatment of CD30+ T cell lymphoma (TCL) prior to the use of CAR-T cells. We hypothesize that TCA cycle inhibition will increase the sensitivity of TCL cells to bendamustine, negatively impact the function of immunosuppressive cell populations that depend on mitochondrial metabolism, and create a conducive environment for enhanced CAR-T cell efficacy. To test our hypothesis, we developed a model of CD30+ TCL, evaluated the efficacy of the combination, and characterized its effects by transcriptome profiling of immune cell populations in tumors. Methods: Mouse TCL cell line EL4-LUC2 was engineered to express human CD30 by retroviral transduction. Single and combination in vitro efficacy was evaluated by viability assays. Combination indices (CI) were calculated using Compusyn. CD30+ EL4-LUC2 cells (EL4.CD30) were inoculated subcutaneously into the flanks of C57Bl6 mice, tumor volumes were calculated, and CD30 expression in tumors was evaluated by immunohistochemical staining and flow cytometry. Transcriptome profiling was performed on a representative subset of tumors by single cell RNA sequencing (scRNA-seq). Results: EL4.CD30 cells displayed comparable CD30 expression to the human TCL cell line Karpas 299. EC 50 values for Devimistat and bendamustine in EL4.CD30 cells were 64.9 mM (95% CI: 62.4-67.4) and 101.2 mM (96.2-106.4), respectively; in Karpas 299, EC 50 values for Devimistat and bendamustine were substantially higher: 117.2 mM (114.7-119.6) and 188.2 mM (180.0 - 196.8), respectively. When dosed in combination, devimistat and bendamustine (D/B) showed synergy (CI = 0.5-0.8) at effect levels > 0.9 in 8 of the 16 dose combinations tested. D/B synergy at an effect level > 0.9 was less overt in Karpas 299 with only 2 of 16 tested combination levels displaying CI values of 0.5-0.9. Efficacy studies with EL4.CD30 tumor-bearing mice revealed potent D/B anti-tumor activity relative to vehicle and single-agent treatment groups. Six complete regressions and 4 partial regressions were observed in the D/B group (Fig. 1A). Moreover, tumor growth rates and median survival values were significantly different (p Conclusions: Our data demonstrate that D/B has potent pharmacological activity in vitro and in vivo and promotes CTL influx into tumors. This suggests that D/B creates an immunopermissive environment fit for CAR-T cell activity. Subsequent studies will investigate the efficacy of CD30.CAR-T cells following D/B treatment. A Phase II pilot study evaluating the feasibility, safety, and tolerability of D/B in patients with relapsed/refractory T-cell Non-Hodgkin Lymphoma is currently ongoing (NCT04217317). Figure 1 Figure 1. Disclosures Pardee: Rafael Pharmaceuticals: Consultancy, Research Funding; Karyopharm Pharmaceuticals: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Pharmacyclics: Speakers Bureau.

BACKGROUND The tumor immune microenvironment (TME) of Glioblastoma consists of almost myeloid-derived macrophages and microglia called TAMs. We have shown that the disruption of CD47-Sirpα-axis induces an antitumor activity of TAMs against GBM in immune-deficient mice, through increases of phagocytosis of tumor cells by TAMs. We have aimed to study the role of microglia and its activation/depletion on GBM progression, in the syngeneic GBM model in immune-competent mice. We have studied the interplay of innate and adaptive immune response after activation and depletion of microglia and the effect on tumor progression and outcome of the mice. MATERIAL AND METHODS We used different colonies of genetically modified immunocompetent mouse strains to genetically activate/deplete microglia in the tumor context. We generated Sall1 CreERT2/fl mice and Cre-negative littermates. The application of Tamoxifen in this constellation leads to the excision of the transcription factor Sall1 and subsequent enhanced microglia activity. Conversely, we generated Sall1 CreERT2 x Csf1r fl/fl animals and the respective heterozygous and Cre-negative littermates in which Tamoxifen treatment leads to inactivation of microglia through the deletion of Csf1r. Glioblastoma tumors were induced by intracerebral injection of GL261, CT2A, or retrovirus-induced PDGF-Akt in pups and Tamoxifen treatment was started once the tumors were detected. RESULTS We observed a survival advantage in tumor-bearing mice after activation of microglia in Sall1 CreERT/fl animals compared to Cre-negative littermates. Genetic depletion of microglia in this model resulted in a shorter lifespan in microglia-depleted animals compared to Cre-negative littermates. Furthermore, the iTME in these tumors is subjected to scRNAseq analysis to identify mechanistic insights. CONCLUSION Microglia are important players in tumor development and progression of glioblastoma in mouse models. These cells may be targeted in future immunotherapeutic approaches for patients.

BackgroundWe previously described ATRC-101, a fully human, engineered IgG1 antibody which is currently under evaluation in the clinic as a monotherapy for solid tumors. A chimeric version of this antibody expressed on a mouse IgG2a (mATRC-101) has shown robust anti-tumor activity as a monotherapy in the EMT6 syngeneic tumor model. In order to assess the potential utility of ATRC-101 in combination with chemotherapeutic agents, non-clinical studies were performed to assess the efficacy of mATRC-101 in combination with chemotherapeutic agents, including Doxorubicin and pegylated liposomal Doxorubicin (PLD), and the impact of these anti-tumor small molecules on mATRC-101 immunoreactivity in mouse tumor and normal tissues.MethodsFemale BALB/c mice with established EMT6 tumors were dosed with mATRC-101 (1 or 3 mg/kg) or vehicle intraperitoneally (IP) twice weekly plus Doxorubicin (2 or 5 mg/Kg) or vehicle (saline) IV once weekly following randomization on Day 6. Statistical analyses of tumor volumes were performed using the normalized area above the curve and the normalized growth rate metrics developed at Atreca. One-sided log-rank (Mantel-Cox) test was used to assess survival advantage relative to the indicated reference group. P-values ≤0.05 were considered significant. In monotherapy studies with Doxorubicin and PLD, EMT6 tumor and non-tumor bearing mice were dosed with vehicle or Doxorubicin (2, 10 mg/kg) or PLD (1, 2, 5,10 mg/kg). Normal mouse tissues were collected at 24 hours and 2 weeks after the last dose. Reactivity for mATRC-101 in EMT6 tumor and normal mouse tissues was evaluated by immunohistochemistryResultsThe combination of 3 mg/kg mATRC-101 and 5 mg/kg Doxorubicin demonstrated significant tumor growth inhibition compared to either monotherapy, or vehicle (pConclusionsThis study provides non-clinical evidence that administration of mATRC-101 in combination with Doxorubicin increases anti-tumor activity in the EMT6 model. Exposure to Doxorubicin or PLD in EMT6 tumor-bearing mice increased mATRC-101 immunoreactivity in the tumor, in a dose-dependent manner Moreover, mATRC-101 immunoreactivity in normal tissues was not influenced by Doxorubicin or PLD. Taken together, these findings support clinical evaluation of the combination of ATRC-101 and doxorubicin.

Exposure to exercise following a breast cancer diagnosis is associated with reductions in the risk of recurrence. However, it is not known whether breast cancers within the same molecular-intrinsic subtype respond differently to exercise. Syngeneic mouse models of claudin-low breast cancer (i.e., EO771, 4TO7, and C3(1)SV40Tag-p16-luc) were allocated to a uniform endurance exercise treatment dose (forced treadmill exercise) or sham-exercise (stationary treadmill). Compared to sham-controls, endurance exercise treatment differentially affected tumor growth rate: 1- slowed (EO771), 2- accelerated (C3(1)SV40Tag-p16-luc), or 3- was not affected (4TO7). Differential sensitivity of the three tumor lines to exercise was paralleled by effects on intratumoral Ki-67, Hif1-α, and metabolic programming. Inhibition of Hif1-α synthesis by the cardiac glycoside, digoxin, completely abrogated exercise-accelerated tumor growth in C3(1)SV40Tag-p16-luc. These results suggest that intratumoral Hif1-α expression is an important determinant of claudin-low breast cancer adaptation to exercise treatment.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/β-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of β-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma., Waaler et al. show that a tankyrase inhibitor, G007-LK, decreases WNT/β-catenin and YAP signaling, sensitizing tumors to anti-PD-1 immune checkpoint therapy in mice. This study suggests that a combinatorial therapy using tankyrase inhibition can be used to overcome β-catenin-mediated resistance to immune checkpoint blockade in melanoma.

Despite tremendous efforts in the last decade to improve treatments, melanoma still represents a major therapeutic challenge and overall survival of patients remains poor. Therefore, identifying new targets to counteract melanoma is needed. In this scenario, autophagy, the "self-eating" process of the cell, has recently arisen as new potential candidate in melanoma. Alongside its role as a recycling mechanism for dysfunctional and damaged cell components, autophagy also clearly sits at a crossroad with metabolism, thereby orchestrating cell proliferation, bioenergetics and metabolic rewiring, all hallmarks of cancer cells. In this regard, autophagy, both in tumor and host, has been flagged as an essential player in melanomagenesis and progression. To pave the way to a better understanding of such a complex interplay, the use of genetically engineered mouse models (GEMMs), as well as syngeneic mouse models, has been undoubtedly crucial. Herein, we will explore the latest discoveries in the field, with particular focus on the potential of these models in unraveling the contribution of autophagy in melanoma, along with the therapeutic advantages that may arise.

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

Transplantation of cardiomyocytes derived from induced pluripotent stem cell (iPSC-CMs) is a promising approach for increasing functional CMs during end-stage heart failure. Although major histocompatibility complex (MHC) class I matching between donor cells and recipient could reduce acquired immune rejection, innate immune responses may have negative effects on transplanted iPSC-CMs. Here, we demonstrated that natural killer cells (NKCs) infiltrated in iPSC-CM transplants even in a syngeneic mouse model. The depletion of NKCs using an anti-NKC antibody rescued transplanted iPSC-CMs, suggesting that iPSC-CMs activated NKC-mediated innate immunity. Surprisingly, iPSC-CMs lost inhibitory MHCs but not activating ligands for NKCs. Re-expression of MHC class I induced by IFN-γ as well as suppression of activating ligands by an antibody rescued the transplanted iPSC-CMs. Thus, NKCs impede the engraftment of transplanted iPSC-CMs because of lost MHC class I, and our results provide a basis for an approach to improve iPSC-CM engraftment.

Synthetic biology is transforming therapeutic paradigms by engineering living cells and microbes to intelligently sense and respond to diseases including inflammation1,2, infections3-5, metabolic disorders6,7, and cancer8,9. However, the ability to rapidly engineer new therapies far outpaces the throughput of animal-based testing regimes, creating a major bottleneck for clinical translation10,11. In vitro approaches to address this challenge have been limited in scalability and broad-applicability. Here, we present a bacteria-in-spheroid co-culture (BSCC) platform that simultaneously tests host species, therapeutic payloads and synthetic gene circuits of engineered bacteria within multicellular spheroids over a timescale of weeks. Long-term monitoring of bacterial dynamics and disease progression enables quantitative comparison of critical therapeutic parameters such as efficacy and biocontainment. Specifically, we screen S. typhimurium strains expressing and delivering a library of antitumor therapeutic molecules via several synthetic gene circuits. We identify novel candidates exhibiting significant tumor reduction and demonstrate high similarity in their efficacies using a syngeneic mouse model. Lastly, we show that our platform can be expanded to dynamically profile diverse microbial species including L. monocytogenes, P. mirabilis, and E. coli in various host cell types. This high-throughput framework may serve to accelerate synthetic biology for clinical applications and understanding the host-microbe interactions in disease sites.

Melanoma is an immunogenic tumor that can metastasize quickly to proximal and distal sites, thus complicating the application of therapeutic modalities. Numerous mouse model systems have been used to gain understanding of the immunobiology and metastatic potential of melanoma. Here, we report the optimization of a syngeneic mouse melanoma model protocol using the mouse B16-derived melanoma cell line B16F10 that ensures the production of tumors on mice pinnae that are similar in size between animals and that enlarge in a time-dependent manner. In this model, B16F10 cells are first allowed to develop tumors after injection in the interscapular area or flank of C57BL/6J mice. Subsequently, the tumors are harvested, cells dissociated and injected into mouse pinnae. Dose-dependent studies revealed that injection of 2 × 10(5) cells allowed for slow tumor enlargement, producing tumors averaging 100 mm(3) within 2 to 3 wk with a metastatic frequency of 100%. This experimental protocol will be useful in dissecting the immunobiology of melanoma tumor development and metastasis and the evaluation of immunotherapeutic antimelanoma therapies.

Introduction: Photoimmunotherapy is an investigational anticancer treatment platform that combines the cell surface binding of an antibody conjugated to a light activatable dye (IRDye® 700DX, IR700) with non-thermal red light illumination for selective cell killing. PD-L1, the target of anti-PD-L1 inhibitors, is a suppressive checkpoint marker expressed on tumor cells and immunosuppressive myeloid cells in the tumor microenvironment. Here we examined the anticancer activity and immune activation elicited by anti-PD-L1-IR700 photoimmunotherapy (PD-L1 photoimmunotherapy). Methods: CT26 or LL/2 tumors were used to assess antitumor immune response following PD-L1 photoimmunotherapy. CT26PD-L1-/- tumors were generated by CRISPR/Cas9. Tumor volume was determined by caliper measurements in illuminated and non-illuminated tumors. Intratumoral immune responses were assessed by flow cytometry. Complete responder (CR) mice were challenged with either CT26 or 4T1 tumors to evaluate immune memory. Results: Mice bearing CT26 or LL/2 tumors treated with PD-L1 photoimmunotherapy exhibited a notable reduction of tumor growth compared to mice that received control treatments (saline, anti-PD-L1-IR700 conjugate alone without illumination, or multi-dosing with anti-PD-L1 antibody). Pre-treatment depletion of CD8+ T cells in the mice abrogated the antitumor activity of PD-L1 photoimmunotherapy, demonstrating a key role of CD8+ T-cell effector activity in the responses. PD-L1 photoimmunotherapy induced CRs in 7/15 mice, and all CR mice rejected CT26 tumor growth after re-challenge, indicating the generation of immunological memory. Furthermore, 6/8 mice rejected inoculation of 4T1 tumors, suggesting an enhanced ability to prime new T cells with tumor neoantigens. Intratumoral immune cell analysis showed a reduction of myeloid cells 2 hours following illumination, an increase of CD103+ dendritic cells 2 days following illumination, and an increase of non-exhausted PD-1-CD8+ T effector cells 8 days after illumination. In a bilateral tumor model, PD-L1 photoimmunotherapy resulted in tumor reduction in the non-illuminated tumor. Mice bearing CT26PD-L1-/- tumor cells similarly exhibited tumor reduction after PD-L1 photoimmunotherapy, suggesting that antitumor activity resulted from the elimination of PD-L1+ non-tumor cells. Conclusions: PD-L1photoimmunotherapy induces anticancer responses by killing PD-L1+ myeloid cells and possibly PD-L1+ cancer cells within the tumor, resulting in augmentation of local and systemic antitumor immunity. These results indicate that PD-L1 photoimmunotherapy can elicit anticancer immune responses in target and distal tumors, including syngeneic mouse models refractory to checkpoint inhibition. Citation Format: Amy H. Thorne, Michelle H. Hsu, Daniel Mendoza, Jason Lapetoda, Christopher M. Parry, Jerry J. Fong, Miguel Garcia-Guzman. PD-L1 photoimmunotherapy kills immunosuppressive myeloid cells to activate local and systemic antitumor immunity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1796.

INTRODUCTION: Preclinical research into immune checkpoint blockade (ICB) requires in vivo models with fully functional immunity. Syngeneic mouse models are homografts derived by transplanting immortalized mouse cancer cell lines or tumor tissues into strain-matched mouse hosts, and are well suited for evaluating the efficacy of new immunotherapies due to retaining an intact mouse immune system. Tumor growth inhibition (TGI) is widely used to characterize drug efficacy, however, a considerable amount of heterogeneity among TGI is often observed for a given ICB treatment in the same syngeneic mouse model. It is of vital importance to understand the source of such variation. The present study sets out to systematically investigate the variation of TGI for ICB treatment in syngeneic mouse models, using tumor growth data of anti-PD-1 treatment in the CT26.WT syngeneic mouse model for 27 independent studies. METHODS: For each study, we calculated TGI and median area under curve (AUC) ratio on the corresponding study day when tumor volume data was collected. We utilized standard deviation and inter quantile range to quantify the variation. We evaluated the relationship between TGI and the corresponding day, and also compared the variation between TGI and median AUC ratio. Furthermore, to better understand the source of drug efficacy variation, we utilized a Bayesian multilevel model to quantify the amount of tumor growth variation at both the study and mouse level. RESULTS: We found that TGI variation was much higher at earlier stages of studies. In contrast, the median AUC ratio showed smaller variation across the duration of each study than TGI for the majority of studies. Additionally, we found neither TGI nor median AUC ratio had significant correlation with the number of mice used in the study. Based on a Bayesian multilevel model, we observed that drug efficacy had larger variation on the mouse level (i.e. within a study), than variation on the study level (i.e. observed between studies), which could indicate heterogeneity of the tumor microenvironment (e.g. TILs) among mice play a major role in the variation of ICB treatment efficacy among studies. CONCLUSIONS: Response to ICB treatment among mice of the same syngeneic model is inherently heterogeneous. TGI of later time points should be favored to reduce variation, and generally median AUC Ratio is more stable than TGI. Citation Format: Binchen Mao, Sheng Guo, Davy Ouyang, Henry Li. Evaluating variation in drug efficacy endpoints in a syngeneic mouse model (CT26.WT) under immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 265.

The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with 1 year median survival. The classical subtype of GBM occurs in about 10% of GBM cases and has the worst clinical outcome. The most frequent genetic alteration associated with classical GBM is amplification in the epidermal growth factor receptor (EGFR) and homozygous deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A). We generated a transgenic mouse harboring EGFRvIII, a constitutively active mutant of EGFR, knock in and homozygous deletion in p19 ARF, an alternative spliced form of the CDKN2a gene, restricted to glial cells. Transgenic mice develop tumors as early as 1 month and 73% of mice die by 6 months of age. MRIs reveal that mice develop infiltrative tumor cells throughout the brain and craniospinal junction with some infiltration within the leptomeninges that can result in hydrocephalus. Histology shows that 72% of tumors are high grade gliomas and some tumors demonstrate classical histological features of human GBM such as pseudopallisading necrosis, hyper vascularity and cellular atypia. We generated primary cell lines from transgenic mice that retained EGFRVIII expression and P19 ARF deletion and formed allografts in syngeneic mouse brains. Epigenetic modifications are considered a key mechanism in GBM development. We have therefore evaluated the efficacy of a panel of 42 compounds that target various epigenetic proteins including the readers, writers, and editors of several histone marks available from the Structural Genomics Consortium in primary cell lines derived from our mouse model. The primary cell lines were exposed to epigenetic drugs showing different responses to the same epigenetic modifiers. Key candidate drugs will further be tested pre-clinically in our syngeneic mouse model. In summary, we developed a novel transgenic and syngeneic mouse model for human classical GBM that can be used for drug screening and pre-clinical trials.

Immunotherapy has potential to improve outcome for cancer patients. The MRI biomarker apparent diffusion co-efficient (ADC) can map response to radiotherapy (RT) through its sensitivity to changes in tumor fluid and cellularity. The role of ADC in evaluating immunotherapy agents is unknown, despite investigators using ADC change as an exploratory endpoint in early phase clinical trials. Our study sought to evaluate and validate ADC changes induced by both RT and immunotherapy agents in syngeneic mouse tumor models. Three experiments were performed in BALB/c mice bearing CT26 colorectal cancers: (1) single 10Gy fraction RT versus control (sham); (2) TLR 7/8 agonist R848 versus control (saline); (3) anti-PD-L1 antibody versus control (saline). We acquired MRI data at 7T Bruker system at days 0, 3, 7, +/- 10 after therapy start, with tumors measuring between 250-300 mm3 at day 0. Median ADC and the inter-quartile range (IQR; a measure of tumor heterogeneity) were derived. Three further equivalent experiments were performed in BALB/c mice bearing triple negative 4T1 breast tumors. All CT26 and 4T1 tumors were bisected at cull for immunohistochemistry and FACS analysis. For CT26 model, RT-induced tumor growth inhibition (p In conclusion, our data are the first to evaluate ADC changes induced by RT and immunotherapy agents in syngeneic mouse models of cancer. RT, R848 and anti-PD-L1 induced different ADC responses, each with varied relationships to immune cells. This highlights the need for extensive validation before diffusion weighted MRI biomarkers can be used in clinical trials to monitor response to immunotherapies alone or in combination with RT. Citation Format: Grazyna Lipowska-Bhalla, Damien J. McHugh, Muhammad Babur, Isabel Peset Martin, Michael Berks, Ross A. Little, Susan Cheung, Yvonne Watson, Denis G. Alferez, Kaye J. Williams, Jamie Honeychurch, James P. O'Connor. Diffusion weighted MRI evaluation of response to immunotherapy and radiotherapy in CT26 and 4T1 syngeneic mouse models of cancer [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 2781.

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. β-catenin is the key transcriptional regulator of WNT/β-catenin signaling. Evidence for β-catenin-mediated immune evasion is found in 13% of all cancers, 42% of primary cutaneous melanoma and a mouse melanoma model. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling to counteract checkpoint inhibitor resistance in melanoma. Here we report that a specific small-molecule tankyrase inhibitor, G007-LK, attenuates WNT/β-catenin and YAP signaling pathways in the syngeneic murine B16-F10 melanoma model enabling sensitivity to anti-PD-1 immune checkpoint therapy. RNA sequencing of 18 tankyrase inhibitor-treated human melanoma cell lines and B16-F10 cells revealed a transcriptional response profile for a subpopulation. This cell line sub-group displayed elevated baseline YAP signaling activity and was susceptible to reduce melanocyte inducing transcription factor (MITF) expression upon tankyrase inhibition.

Syngeneic mouse models enable us to study the interaction of the immune system with tumor cells during tumor development and metastasis. It further allows us to evaluate the efficacy of immunotherapies on the different stages of tumor growth and the metastatic process. Here we describe two syngeneic mouse models, a murine mammary carcinoma and a murine melanoma model, that are used to study metastasis. Metastases occur spontaneously in the murine mammary carcinoma. The presence and number of foci are evaluated by culturing the lung tissue in a colony formation assay. For the murine melanoma model, tumor cells are injected intravenously, and metastatic burden is analyzed by counting of metastatic lesions.

FoxP3+ regulatory T cells (Treg) are essential to prevent autoimmunity but have also been implicated in suppressing antitumor immune responses. We assessed the impact of depletion of Treg cells in 8 different syngeneic tumor models, including cancer immunotherapy-sensitive and -resistant models. Robust, rapid tumor control or regression was observed in all models tested. Analysis of both peripheral and tumor-infiltrating T cells revealed that Treg cell depletion resulted in robust proliferation and cytokine production by CD8+ T cells in lymph nodes, spleen and tumor tissue. Tumor regression was CD8+ T cell-dependent, as antibody-mediated depletion of these cells abrogated tumor regression in a representative model. Interestingly, studies using FTY720 to pharmacologically block T-cell egress from the lymph nodes suggest that following Treg depletion cancer immunotherapy-sensitive models may not require new T-cell infiltration into tumor for tumor regression. This is in contrast to an immunotherapy-resistant model, which failed to regress following Treg depletion when lymph node egress was concurrently blocked. These data suggest that Treg cells play a critical role in suppressing both priming of novel anti-tumor CD8+ T-cell responses as well as limiting effector functionality of tumor-infiltrating CD8+ T cells within the tumor microenvironment. Citation Format: Yingyun Wang, Teresita Delfino, Jill M. Schartner, Sascha Rutz. Targeted ablation of FoxP3+ T cells activates peripheral and tumor-infiltrating cytotoxic CD8+ T cells in multiple syngeneic mouse 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 B94.

The mechanisms that influence metastatic growth rates are poorly understood. One mechanism of interest known as concomitant tumour resistance (CTR) can be defined as the inhibition of metastasis by existing tumour mass. Conversely, the presence of a primary tumour has also been shown to increase metastatic outgrowth, termed concomitant tumour enhancement (CTE). The majority of studies evaluating CTR/CTE in preclinical models have relied on endpoint histological evaluation of tumour burden. The goal of this research was to use conventional magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging to study the impact of a primary tumour on the development of brain metastases in a syngeneic mouse model. Here, we report that the presence of a 4T1 primary tumour significantly enhances total brain tumour burden in Balb/C mice. Using in vivo BLI/MRI we could determine this was not related to differences in initial arrest or clearance of viable cells in the brain, which suggests that the presence of a primary tumour can increase the proliferative growth of brain metastases in this model. The continued application of our longitudinal cellular and molecular imaging tools will yield a better understanding of the mechanism(s) by which this physiological inhibition (CTR) and/or enhancement (CTE) occurs.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Background 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). Methods 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. Results 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. Conclusions 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.

Background Patient outcomes with pancreatic ductal adenocarcinoma (PDA) remain dismal, despite the introduction of combination chemotherapies. Response to therapy is hampered by tumour stroma, which is highly heterogeneous but poorly characterised. The clinical success of immunotherapy in certain cancer types justifies investigation in PDA using a personalised approach of selecting pre-clinical models. Methods Patient PDA samples (n = 70; International Cancer Genome Consortium) were subjected to hierarchical clustering using a gene set representing CD8 and regulatory T cells to identify sub-groups. Syngeneic mouse models recapitulating these sub-groups (cross-species analysis) were developed using cell lines from genetically engineered mouse (GEM) models. Models bearing subcutaneous tumours were treated with anti-GITR agonist (DTA-1; 10mg/kg) or isotype (IgG2b) and tissues were collected for 12-color fluorescence activated cell sorting (FACS), NanoString and immunohistochemistry (IHC). Results Using clustering analysis, we identified four distinct patient PDA immune sub-groups with differential expression of T cell genes. These sub-groups showed differential expression of glucocorticoid-induced TNF receptor (GITR) and its associated genes. Hence, we hypothesised that these groups may respond differently to anti-GITR therapy in vivo. As expected, the three syngeneic models showed differential response to anti-GITR treatment. Model with increased GITR expression (>2-fold; NanoString and immunoblot) showed a significant (p = 0.02) survival benefit and a modest decrease in tumour burden upon anti-GITR treatment (n = 10) compared to the isotype control (n = 7) and the other two models. We observed anti-GITR mediated increase in GITR+ CD8+ cells and no change in GITR+ FOXP3+ cells (FACS, NanoString and IHC) along with increased gene expression of Pdcd1, Lag3, Spn and Itk. Conclusion Our models recapitulate the immunological gene expression profiles observed in patient PDA samples making them a highly useful resource to study personalized immunotherapies. This integrated study highlights the importance of patient selection to maximize therapy benefit and spare immune related adverse events. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure D. Cunningham: Research grant / Funding (self): Amgen; Research grant / Funding (self): Sanofi; Research grant / Funding (self): Merrimack; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Celgene; Research grant / Funding (self): MedImmune; Research grant / Funding (self): Bayer; Research grant / Funding (self): 4SC; Research grant / Funding (self): Clovis; Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): Janssen; Research grant / Funding (self): Merck. N. Starling: Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Pfizer; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Eli Lilly; Travel / Accommodation / Expenses: Merck; Travel / Accommodation / Expenses: Roche; Honoraria (self): Eli Lilly; Honoraria (self): Merck Serono; Honoraria (self): MSD Oncology; Honoraria (self): Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Servier. A. Sadanandam: Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Merck KGaa; Research grant / Funding (self): Pierre Fabre. All other authors have declared no conflicts of interest.

BACKGROUND Checkpoint inhibitors as well as adoptive cell therapy hold great promise for cancer treatment and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. MATERIALS AND METHODS The murine glioma cell line GL261 was transfected with HER2. Tumor cells were orthotopically implanted into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined, lymphocyte infiltration and immunosuppressive TME were characterized. RESULTS Combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody resulted in synergistic tumor regression and long-term survival of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells. CONCLUSION These data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced by co-therapy with checkpoint inhibitors, resulting in successful treatment of advanced tumors refractory to mono-therapy. Furthermore, this combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are planning a phase I clinical study (CAR2BRAIN-CHECK).

MDM2-p53 antagonists block the interaction between the Tumor Protein p53 and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2-p53 antagonists are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2-p53 antagonists are currently being evaluated in early clinical development. BI-907828 is a novel and potent MDM2-p53 antagonist with optimized drug-like properties that has shown efficacy in human tumor xenograft models at daily low oral dose as well as intermittent high dose schedules. Recent preclinical studies in syngeneic mouse models of cancer have demonstrated that BI-907828, apart from its direct tumor-targeting activity, also has immunomodulatory activity shown to contribute to efficacy. Single-agent BI-907828 induced anti-tumor immunological memory that controlled tumor growth in a re-challenge study. Moreover, a dual combination with an anti-mouse PD-1 checkpoint inhibitor resulted in synergistic efficacy in a syngeneic mouse model of cancer (AACR 2018, abstract 4866). Here we present data for the triple combination of BI-907828 with two checkpoint inhibitors. In syngeneic mouse tumor models (Colon-26 and B16-F10), the combination of BI-907828 with tool antibodies against mouse PD-1 and mouse LAG-3 shows high response rates of 50-90% with tumor regressions observed for even very large tumors. Moreover, efficacy of the triple combination is superior to each single agent and all dual combinations. An antibody-mediated depletion study suggests a contribution of CD8+ T cells but not of CD4+ T cells to full efficacy of the triple combination. FACS analysis of tumors isolated from Colon-26 tumor-bearing mice indicates that treatment with the triple combination leads to expansion of tumor-infiltrating CD8+ T cells. In summary, BI-907828 is a novel, potent, orally bioavailable MDM2-p53 antagonist that shows synergistic efficacy in a triple combination with antibodies targeting the immune checkpoints PD-1 and LAG-3 in syngeneic mouse models of cancer. BI-907828 is currently under evaluation in a Phase I clinical study (NCT03449381). Citation Format: Dorothea Rudolph, Ulrike Weyer-Czernilofsky, Markus Reschke, Martina Sykora, Jörg Rinnenthal, Sophia Blake, Gabriela Gremel, Andreas Wernitznig, Andreas Gollner, Norbert Kraut, Jürgen Moll. BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of 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 3197.

Metallic nanorods are promising agents for a wide range of biomedical applications. We report an optical hyperthermia method capable of inducing slowdown tumor progression of an experimental in vivo CT-2A glioblastoma tumor. The tumor model used in this research is based on the transplantation of mouse astrocytoma CT-2A cells in the striatum of mice by intracranial stereotaxic surgery. Two weeks after cell implant, the resulting tumor is treated by irradiating intratumoral injected gold nanorods, biofunctionalized with CD133 antibody (B-GNRs), using a continuous wave laser. Nanoparticles convert the absorbed light into localized heat (reaching up to 44 °C) due to the effect of surface plasmon resonance. A significant slowdown in CT-2A tumor progression is evident, by histology and magnetic resonance imaging, at one (p = 0.03) and two weeks (p = 0.008) after irradiation treatment. A notable deceleration in tumor size (15%-75%) as compared to the control untreated groups, it is observed. Thus, laser irradiation of B-GNRs is found to be effective for the treatment of CT-2A tumor progression. Similarities between the pre-clinical CT-2A tumor model and the human astrocytoma disease, in terms of anatomy, metastatic behavior and histopathology, suggest that hyperthermic treatment by laser irradiation of B-GNRs administered into high-grade human astrocytoma might constitute a promising alternative treatment to limit the progression of this deadly disease., We thank Ms Soledad Martinez (cell culture experiments), Centre for Biomedical Technology (CTB) for technical help. Also, PhD Teresa López de Mora for critical reading of the manuscript. The authors are thankful to their grant supporters, Universidad Nacional Experimental del Táchira (UNET)— Venezuela to Oscar Casanova-Carvajal. This study was also financially supported in part by CIBER-BBN (Spain) and NEUROCENTRO-CM (B2017/BMD-3760) consortium. Characterization of the MNPs has been performed by the ICTS 'NANBIOSIS', Unit 15 (Functional Characterization of Magnetic Nanoparticles) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the Center for Biomedical Technology (CTB) of the 'Universidad Politécnica de Madrid' (UPM). This work was carried out as part of Project PGC2018-097531-B-I00, funded by the Ministry of Science of Spain.

e17078 Background: Alterations in the Wnt/β-catenin pathway have been associated with tumor progression in multiple cancers. Gene expression profiling in ovarian cancer has correlated Wnt upregulation to patterns of immune evasion in the tumor microenvironment (TME). We investigated effects of in vitro Wnt inhibition in a murine ovarian cancer cell line with p53 knocked out as a model to mimic human ovarian carcinoma. Methods: We used a small molecule inhibitor, CGX-1321, in C57Bl6 mice, an immunocompetent mouse model, injected intraperitoneally with either ID8 cells, a murine ovarian cancer cell line, or ID8-p53-/- cells. Mouse omentums were collected and weighed for evaluation of tumor growth. Representative histology slides were stained for H&E. Western blots for β-catenin baseline levels were performed on the cell lines. TopFlash Assays with WNT3A stimulation were used for analysis of cell line nuclear β-catenin levels. Flow cytometry was performed on mouse omentums for TME evaluation. Results: Treatment with CGX-1321 decreased tumor size via omentum weight with both cell lines, p = 0.0098 with ID8 cells and p = 0.0855 with ID8-p53-/- cells. H&E slides revealed a decreased tumor progression. Western blots confirmed a difference in baseline β-catenin levels between these two cell lines. However, TopFlash assays showed a response to stimulation. Changes in the TME between treated samples with ID8-p53-/- cells did now show significant differences via flow cytometry. Conclusions: In the syngeneic mouse model with a murine ovarian cancer cell line, with p53 knocked out, tumor size and proliferation were decreased with treatment with Wnt inhibition. Surprisingly, the TME changes were inconclusive via flow cytometry. Perhaps these cells do not rely upon the Wnt/ β-catenin pathway as heavily, as seen in changes to baseline β-catenin levels, or perhaps tumors progress quicker and the critical time point of TME change is being overlooked. More investigation needs to be performed to further elucidate these differences.