Orthotopic Mouse Research Articles

BET protein inhibition evidently enhances sensitivity to PI3K/mTOR dual inhibition in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC), the second most common primary liver cancer, is a fatal malignancy with a poor prognosis and only very limited therapeutic options. Although molecular targeted therapy is emerged as a promising treatment strategy, resistance to molecular-targeted therapy occurs inevitably, which represents a major clinical challenge. In this study, we confirmed that mammalian target of rapamycin (mTOR) signaling is the most significantly affected pathways in ICC. As a novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, exerts antitumour activity by effectively and specifically blocking the dysfunctional activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. We generate the orthotopic ICC mouse model through hydrodynamic transfection of AKT and yes-associated protein (YAP) plasmids into the mouse liver. Our study confirmed that BEZ235 can suppress the proliferation, invasion and colony conformation abilities of ICC cells in vitro but cannot effectively inhibit ICC progression in vivo. Inhibition of PI3K/mTOR allowed upregulation of c-Myc and YAP through suppressed the phosphorylation of LATS1. It would be a novel mechanism that mediated resistance to PI3K/mTOR dual inhibitor. However, Bromo- and extraterminal domain (BET) inhibition by JQ1 downregulates c-Myc and YAP transcription, which could enhance the efficacy of PI3K/mTOR inhibitors. The efficacy results of combination therapy exhibited effective treatment on ICC in vitro and in vivo. Our data further confirmed that the combination of PI3K/mTOR dual inhibitor and BET inhibition induces M1 polarization and suppresses M2 polarization in macrophages by regulating the expression of HIF-1α. Our study provides a novel and efficient therapeutic strategy in treating primary ICC.

Apatinib inhibits glioma cell malignancy in patient-derived orthotopic xenograft mouse model by targeting thrombospondin 1/myosin heavy chain 9 axis

We determined the antitumor mechanism of apatinib in glioma using a patient-derived orthotopic xenograft (PDOX) glioma mouse model and glioblastoma (GBM) cell lines. The PDOX mouse model was established using tumor tissues from two glioma patients via single-cell injections. Sixteen mice were successfully modeled and randomly divided into two equal groups (n = 8/group): apatinib and normal control. Survival analysis and in vivo imaging was performed to determine the effect of apatinib on glioma proliferation in vivo. Candidate genes in GBM cells that may be affected by apatinib treatment were screened using RNA-sequencing coupled with quantitative mass spectrometry, data mining of The Cancer Genome Atlas, and Chinese Glioma Genome Atlas databases, and immunohistochemistry analysis of clinical high-grade glioma pathology samples. Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and co-immunoprecipitation (co-IP) were performed to assess gene expression and the apatinib-mediated effect on glioma cell malignancy. Apatinib inhibited the proliferation and malignancy of glioma cells in vivo and in vitro. Thrombospondin 1 (THBS1) was identified as a potential target of apatinib that lead to inhibited glioma cell proliferation. Apatinib-mediated THBS1 downregulation in glioma cells was confirmed by qPCR and western blotting. Co-IP and mass spectrometry analysis revealed that THBS1 could interact with myosin heavy chain 9 (MYH9) in glioma cells. Simultaneous THBS1 overexpression and MYH9 knockdown suppressed glioma cell invasion and migration. These data suggest that apatinib targets THBS1 in glioma cells, potentially via MYH9, to inhibit glioma cell malignancy and may provide novel targets for glioma therapy.

Feasibility and utility of MRI and dynamic 18F-FDG-PET in an orthotopic organoid-based patient-derived mouse model of endometrial cancer

BackgroundPelvic magnetic resonance imaging (MRI) and whole-body positron emission tomography-computed tomography (PET-CT) play an important role at primary diagnostic work-up and in detecting recurrent disease in endometrial cancer (EC) patients, however the preclinical use of these imaging methods is currently limited. We demonstrate the feasibility and utility of MRI and dynamic 18F-fluorodeoxyglucose (FDG)-PET imaging for monitoring tumor progression and assessing chemotherapy response in an orthotopic organoid-based patient-derived xenograft (O-PDX) mouse model of EC.Methods18 O-PDX mice (grade 3 endometrioid EC, stage IIIC1), selectively underwent weekly T2-weighted MRI (total scans = 32), diffusion-weighted MRI (DWI) (total scans = 9) and dynamic 18F-FDG-PET (total scans = 26) during tumor progression. MRI tumor volumes (vMRI), tumor apparent diffusion coefficient values (ADCmean) and metabolic tumor parameters from 18F-FDG-PET including maximum and mean standard uptake values (SUVmax/SUVmean), metabolic tumor volume (MTV), total lesion glycolysis (TLG) and metabolic rate of 18F-FDG (MRFDG) were calculated. Further, nine mice were included in a chemotherapy treatment study (treatment; n = 5, controls; n = 4) and tumor ADCmean-values were compared to changes in vMRI and cellular density from histology at endpoint. A Mann–Whitney test was used to evaluate differences between groups.ResultsTumors with large tumor volumes (vMRI) had higher metabolic activity (MTV and TLG) in a clear linear relationship (r2 = 0.92 and 0.89, respectively). Non-invasive calculation of MRFDG from dynamic 18F-FDG-PET (mean MRFDG = 0.39 μmol/min) was feasible using an image-derived input function. Treated mice had higher tumor ADCmean (p = 0.03), lower vMRI (p = 0.03) and tumor cellular density (p = 0.02) than non-treated mice, all indicating treatment response.ConclusionPreclinical imaging mirroring clinical imaging methods in EC is highly feasible for monitoring tumor progression and treatment response in the present orthotopic organoid mouse model.

Development of theranostic dual-layered Au-liposome for effective tumor targeting and photothermal therapy

BackgroundPhotothermal therapy (PTT) is an emerging anti-cancer therapeutic strategy that generates hyperthermia to ablate cancer cells under laser irradiation. Gold (Au) coated liposome (AL) was reported as an effective PTT agent with good biocompatibility and excretory property. However, exposed Au components on liposomes can cause instability in vivo and difficulty in further functionalization.ResultsHerein, we developed a theranostic dual-layered nanomaterial by adding liposomal layer to AL (LAL), followed by attaching polyethylene glycol (PEG) and radiolabeling. Functionalization with PEG improves the in vivo stability of LAL, and radioisotope labeling enables in vivo imaging of LAL. Functionalized LAL is stable in physiological conditions, and 64Cu labeled LAL (64Cu-LAL) shows a sufficient blood circulation property and an effective tumor targeting ability of 16.4%ID g−1 from in vivo positron emission tomography (PET) imaging. Also, intravenously injected LAL shows higher tumor targeting, temperature elevation in vivo, and better PTT effect in orthotopic breast cancer mouse model compared to AL. The tumor growth inhibition rate of LAL was 3.9-fold higher than AL.ConclusionBased on these high stability, in vivo imaging ability, and tumor targeting efficiency, LAL could be a promising theranostic PTT agent.Graphic

Biomolecular imaging of colorectal tumor lesions using a FITC-labeled scFv-C\u03ba fragment antibody

For the sensitive diagnosis of colorectal cancer lesions, advanced molecular imaging techniques using cancer-specific targets have emerged. However, issues regarding the clearance of unbound probes and immunogenicity remain unresolved. To overcome these limitations, we developed a small-sized scFv antibody fragment conjugated with FITC for the real-time detection of colorectal cancer by in vivo molecular endoscopy imaging. A small-sized scFv fragment can target colon cancer secreted protein-2 (CCSP-2), highly expressed in colorectal adenocarcinoma tissues; moreover, its full-length IgG probe has been used for molecular imaging previously. To assess the efficacy of anti-CCSP-2 scFv-FITC, surgical specimens were obtained from 21 patients with colorectal cancer for ex vivo molecular fluorescence analysis, histology, and immunohistochemistry. Orthotopic mice were administered with anti-CCSP-2 scFv-FITC topically and intravenously, and distinct tumor lesions were observed by real-time fluorescence colonoscopy. The fluorescence imaging of human colon cancer specimens allowed the differentiation of malignant tissues from non-malignant tissues (p < 0.05), and the CCSP-2 expression level was found to be correlated with the fluorescence intensity. Here, we demonstrated the feasibility and safety of anti-CCSP-2 scFv-FITC for molecular imaging as well as its potential in real-time fluorescence colonoscopy for the differential diagnosis of tumor lesions.

Abstract 2797: Changes in the phenotype of macrophages and CD8+ T Cells in the perivascular niche of prostate tumours following androgen deprivation

Abstract Men with intermediate/high-risk, localized prostate cancer usually receive neoadjuvant androgen deprivation therapy (ADT). Although this markedly reduces tumor burden, it may not be curative and over time cancer cells develop resistance to ADT causing the tumor to regrow and spreads to other sites like the bones. Tumors are then termed ‘castration resistant' (CR) and require more aggressive forms of treatment like radical prostatectomy (RP) and chemotherapy. These can have unpleasant side effects so preventing the development of castration resistance and subsequent tumor regrowth/metastasis, would be highly beneficial for patients. A distinct subset of perivascular (PV) tumor-associated macrophages (TAMs) expressing the cell-surface receptor, MRC1, has been shown to be proangiogenic, immunosuppressive and to stimulate tumor regrowth after chemotherapy, irradiation or vascular targeting drugs. In the current study, we have examined the density and phenotype of TAMs in PV vs non-PV areas of localized, human prostate tumors and orthotopic mouse (Myc-CaP) tumors following neoadjuvant ADT or castration respectively. In human tumors, MRC1+ TAMs accumulated at highest density around stromal blood vessels, and were abundant in these sites following ADT. In mice, castration markedly reduced primary tumor growth and changed the distribution and phenotype of TAMs and cytotoxic CD8+ T lymphocytes (CTLs) just prior to the development of CR. In PV areas of tumors in sham-castrated (i.e. control) mice there were significantly more TAMs expressing MRC1 and the broad-spectrum negative checkpoint regulator, VISTA than in non-PV areas. This was accompanied by the preferential accumulation of CTLs around blood vessels (contrasting with the even distribution of CD4+ (helper) cells and CD4+FOXP3+ Tregs across PV and non-PV areas). Following castration, the PV density of MRC1+ and VISTA+ TAMs was markedly increased, along with that of CTLs expressing the cytotoxic protease, granzyme B - a marker of active CTLs. Although these cells may cross the vasculature into tumors in an active state, passing through a PV niche enriched in VISTA+ TAMs would likely suppress their cytotoxic function before they progressed into the tumor mass. An interesting picture is, therefore, emerging of tumor-promoting TAMs accumulating around blood vessels after various forms of treatment, where they regulate a number of important events including the extravasation and function of various immune effectors. Citation Format: Haider Al-janabi, Xuan Pu, Munitta Muthana, Ning Wang, Mateus Crespo, Bora Gurel, Johann De Bono, Janet E. Brown, Claire E. Lewis. Changes in the phenotype of macrophages and CD8+ T Cells in the perivascular niche of prostate tumours following androgen deprivation [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 2797.

Abstract 2339: Halting head &amp; neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 14,600 deaths each year in the United States alone. HNSCC is associated with human papillomavirus (HPV) infection, and tobacco use and abusive alcohol intake. Recent revolutionary immunotherapy approaches have changed the landscape of treatment options in HNSCC. However, less than 20% of HNSCC patients respond to FDA-approved anti-PD1 immune checkpoint blockade (ICB) (pembrolizumab and nivolumab), often not leading to durable responses. This highlights the unmet need to identify novel therapeutic options and biomarkers predicting more favorable response to maximize the efficacy of immune-oncology (IO) strategies for HNSCC treatment. Glutamine is a conditionally essential amino acid for rapidly proliferating cancer cells making glutamine pathway inhibition an attractive approach for anti-cancer therapy. Treatment with the glutamine antagonist (DRP-104), which irreversibly inhibits all known enzymes involved in glutamine metabolism within the tumor, resulted in metabolically halted cell growth in vitro in a large panel of cell lines (n=8) representing the spectra of HPV- and HPV+ HNSCC, with an IC50 of 0.2-25uM, Interestingly, HNSCC cells bearing genetic alterations in PIK3CA/PTEN were significantly more sensitive to glutamine antagonism in comparison to unaltered HNSCC cells. The dependence of glutamine in HNSCC growth and the increased sensitivity of PIK3CA/PTEN aberrant cells was also observed in orosphere assays and 6 distinct HNSCC tumor xenografts in NOD SCID mice. We next investigated the efficacy of glutamine suppression in the context of an intact immune system, taking advantage of new syngeneic HNSCC model system, 4MOSC and MOC1. Specifically, we recently developed orthotopic syngeneic mouse models, 4MOSC, reflecting human tobacco-related HNSCC genomic alterations and mutational landscape (Wang et al., Nature Comm 2019), while MOC1 is a DMBA-induced mouse HNSCC cell line grown in the subcutaneous flank of mice (Judd et al. Cancer Res 2011). Monotherapy administration of DRP- 104 (1.4mg/kg) was potent as a monotherapy in all three syngeneic HNSCC models. We also observed a trend of increased sensitivity when DRP-104 was combined with anti-PD-1 therapy. Our data suggest that broad glutamine antagonism using DRP-104 has therapeutic potential in HNSCC by both dismantling cancer metabolism and enhancing the anti-tumor immune response of immune checkpoint blockade. Clinical trials of DRP-104 are currently ongoing. Citation Format: Michael Allevato, Yumi Yokoyama, Robert Wild, Silvio Gutkind. Halting head &amp; neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways [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 2339.

Abstract 86: O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Abstract Cancer cells alter their metabolism to increase cell growth. A subset of the glucose taken up is shunted into the hexosamine biosynthetic pathway where it is used to synthesize UDP-GlcNAc, a substrate of O-GlcNAc transferase (OGT), which modifies cytoplasmic and nuclear proteins with O-linked sugar moieties. Here, we show that OGT and O-GlcNAcylation are elevated in glioblastoma (GBM) cancer cells and in GBM patient samples that correlates with disease progression. Reduction of OGT expression in GBM cells led to significant reduction in anchorage-independent growth, acetyl-CoA levels, and decrease in free fatty acids. Conversely, overexpressing OGT in GBM cells had the opposite effect. Reducing OGT expression in GBMcells transplanted in an orthotopic intracranial mouse model reduced tumor growth and extended survival. Mechanistically, we show that OGT overexpression increases carbon-flux of acetate to acetyl-CoA, a reaction carried by the enzyme acetyl-CoA synthetase 2 (ACSS2). Indeed, OGT regulates ACSS2 protein levels and O-GlcNAcylation increases ACSS2phosphorylation on Ser-267 in a cyclin dependent kinase 5 (CDK5)-dependent manner, which regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Using an ex vivo GBM brain slice model we show that treatment of GBM-transplanted slices with OGT inhibitor Ac-GlcNAc-5S or pan-cdk inhibitor dinaciclib reduced growth of pre-formed tumors in cultured brain slices. These results suggest a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate acetate metabolism and identify OGT and CDK5 as novel therapeutic targets for treatment of glioblastoma. Citation Format: Lorela Ciraku, Zachary Bacigalupa, Rebecca Moeller, Jing Ju, Rusia H. Lee, Christina Ferrer, Sophie Trefely, Nathaniel W. Snyder, Luca D'Agostino, Christos D. Katsetos, Wenyin Shi, Mauricio J. Reginato. O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation [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 86.

Abstract 2010: Pancreatic cancer stem cell exosomes drive a hierarchical intratumor communication network that fuels disease progression through hippo pathway inhibition

Abstract Intra-tumor heterogeneity represents a major challenge for cancer treatment. Resistance to therapy and tumorigenic ability varies greatly between distinct subpopulations of cancer cells. Intercellular communication is critical to establish a cooperative environment that maintains tumor heterogeneity and potentiates disease progression. However, how subpopulations of cancer cells interact to support tumor progression is not fully understood. Exosomes have the potential to re-educate recipient cells through the delivery of their cargo, emerging as the main mediators of intercellular communication. Here we demonstrate that subpopulations of pancreatic cancer cells use exosomes to establish a hierarchical and dynamic communication network, the ExoNet. Exosomes from cancer stem cells (CSC) are preferentially taken up by non-CSC (NCSC). Targeted inhibition of CSC exosomes in PDAC orthotopic, genetically engineered and patient-derived xenograft mouse models is sufficient to impair tumor growth and metastasis establishment. A comprehensive comparative proteomic analysis of exosomes derived from distinct subpopulations of pancreatic ductal adenocarcinoma (PDAC) human cell lines identified a significant enrichment of Agrin in CSC exosomes. Blocking exosomes secretion and downregulating Agrin in CSCs significantly diminishes YAP nuclear localization in receiving cells, indicating that CSC exosomes contribute for Hippo pathway inactivation. Agrin+ circulating exosomes correlate with tumor burden in PDAC patients, and most importantly correlate with CD133+ circulating exosomes, indicating that circulating Agrin+ exosomes are from CSC origin. Longitudinal analysis of circulating Agrin+ exosomes in PDAC patients predicts disease progression and response to therapy. Our study describes a novel role of cancer exosomes in the interplay between subpopulations of cancer cells promoting tumor progression. We identified Agrin as a potential non-invasive biomarker to monitor disease progression and predict treatment response. Citation Format: Carolina F. Ruivo, Nuno Bastos, Ines Batista, Barbara Adem, Cecilia Duraes, Carlos A. Melo, Francisco J. Campos-Laborie, Pedro Moutinho-Ribeiro, Barbara Mourão, Marília Cravo, Guilherme Macedo, Fátima Carneiro, Tony Kouzarides, Raghu Kalluri, Jose C. Machado, Sonia Melo. Pancreatic cancer stem cell exosomes drive a hierarchical intratumor communication network that fuels disease progression through hippo pathway inhibition [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 2010.

Abstract 1785: Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases viral replication and diminishes cell proliferation and induces apoptosis of surrounding effector T-cells. Furthermore, kynurenine (kyn), a metabolite of tryptophan, induces T-cell differentiation into Tregs. Excess kynurenine also elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, D24-RGDOX, clinically known as DNX-2440, triggers IFNγ production contributing to a positive IDO-Kyn-AhR feedback loop. Thus, we hypothesized that combining D24-RGDOX with IDO inhibitors will synergize to treat glioblastoma. We studied the IDO-Kyn-AhR cascade in response to D24-RGDOX infection in human and murine glioblastoma cells. Orthotopic syngeneic glioblastoma mouse models were used to determine efficacy and tumor immune effects of D24-RGDOX and IDO inhibitors as single agents or as a combination treatment. We characterized IDO and AhR in D24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo compared to mock infected controls. D24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. Furthermore, we showed that viral protein expression and viral activity was enhanced in D24-RGDOX infected cultures under IDO inhibition. To test the therapeutic efficacy of combining D24-RGDOX with IDO inhibitors, we used a murine immunocompetent glioblastoma model; the combination produced 30% more long-term survivors compared D24-RGDOX alone (P=0.03, log-rank test). Next, lymphocytic depletion studies illustrated that this anticancer effect was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to D24-RGDOX-treated mice (P=0.009, student t-test). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and MDSCs compared to D24-RGDOX alone (P≤0.05 in all populations, student t-test). This microenvironment remodeling correlated with complete tumor elimination. Altogether, D24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Derek A. Wainwright, Sagar Sohoni, Sumit Gupta, Ashley Ossimetha, Frederick Lang, Marta Alonso, Candelaria Gomez-Manzano, Juan Fueyo. Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation [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 1785.

Abstract 443: Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor models

Abstract The targeting of pathways that regulate tumor angiogenesis is an effective strategy for the treatment of solid tumors. Transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) are master regulators of tumor angiogenesis that limit the efficacy of cancer therapies and suppress the anti-tumor immune response. Combination therapies consisting of immune checkpoint inhibitors and tumor angiogenesis inhibitors are emerging as effective treatments for solid cancers. Bintrafusp alfa (M7824) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 antibody blocking PD-L1. Here, we tested the antitumor efficacy of a combination therapy consisting of bintrafusp alfa plus a VEGF receptor kinase inhibitor (pazopanib, lenvatinib, regorafenib, axitinib, or nintedanib), or a VEGF neutralizing antibody (B20) in multiple preclinical tumor models. The combination of bintrafusp alfa with lenvatinib, regorafenib, axitinib, or nintedanib showed significantly greater antitumor efficacy than the respective monotherapies in the CT26 mouse syngeneic colon carcinoma model. In the 4T1 mouse breast carcinoma model, the combination of bintrafusp alfa with anti-VEGF, lenvatinib, or regorafenib was significantly more efficacious than the respective monotherapies. Additionally, the combination of bintrafusp alfa with pazopanib also showed superior antitumor efficacy in an orthotopic Renca mouse tumor model. These data indicate that bintrafusp alfa may be a viable combination partner with multiple angiogenesis inhibitors and provide a rational for the continued development of these combination therapies to treat solid cancers. Citation Format: Tsz-Lun Yeung, Adam Lazorchak, Huakui Yu, Jin Qi, Guozhong Qin, Bo Marelli, Kai Fu, Yan Lan. Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor 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 443.

Abstract 965: The role of LOX-mediated FAK phosphorylation in osteosarcoma metastasis

Abstract Despite the use of chemotherapy and surgery, the development of metastasis remains to be the leading cause of death in patients with osteosarcoma. LOX encodes for lysyl oxidase, which is an enigmatic molecule in cancer by acting as a tumor suppressor in certain tumors, but a metastatic promoter in other tumors. To investigate the function of LOX, we generated LOX overexpression mutants in two osteosarcoma cell lines and showed that higher LOX expression promoted tumor cell proliferation and invasiveness. Using an orthotopic xenograft mouse model, we further showed that osteosarcoma cells with higher LOX expression produced a higher number of mice with pulmonary metastases than the parental cell line (n=9). To characterize the mechanism of LOX-mediated metastasis, we examined the protein expression of a panel of potential downstream targets. The results demonstrated that phospho-FAK (focal adhesion kinase) expression was increased in the LOX-overexpression mutant relative to the parental cell line. Using a small molecule inhibitor of FAK, we showed that it significantly reduced the LOX-mediated metastasis in the orthotopic xenograft model. To demonstrate the clinical significance of our findings, we analyzed the expression profiling and RNAseq datasets recently generated from the TARGET osteosarcoma study. A Kaplan-Meier analysis showed that a higher level of LOX expression significantly correlated with a poor outcome (p &amp;lt; 0.05) and a multivariate analysis indicated LOX remained to be significant after controlling for initial metastatic status. In addition, we have previously shown that p27 is frequently mislocalized in the cytoplasm of osteosarcoma cells and drives the development of metastasis through interacting with the PAK1-mediated actin cytoskeleton network. Our results showed that the expression of LOX was upregulated in p27-mislocalized osteosarcoma cell lines with high metastatic potential. A tissue microarray analysis showed that cytoplasmic p27 expression significantly correlated with LOX expression (p = 0.003). Together, our findings suggest that a novel link between p27 mislocalization and LOX expression. LOX plays a pivotal role in osteosarcoma metastasis by upregulating FAK phosphorylation. FAK inhibition is a potential therapeutic strategy to reduce the development of metastasis in osteosarcoma with LOX overexpression. Citation Format: Xiang Chen, John M. Hicks, Poonam Sarkar, Waleed M. Gaber, Tsz-Kwong MAN. The role of LOX-mediated FAK phosphorylation in osteosarcoma metastasis [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 965.

Abstract 1229: Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death

Abstract ADP-ribosylation factor 1 (Arf1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, the molecular mechanisms underlying Arf1-dependent cancer development and progression are far from understood. We report here that Arf1 is the most amplified gene in Arf gene family in breast cancer, and knocking it down leads to an induction of autophagy via the AMPK pathway. To effectively target Arf1, we rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides, which can block Arf1 activation more efficiently than other commercially available Arf1 inhibitors through targeting the Arf1-GDP/ARNO complex at the Golgi. The anticancer effects of these γ-dipeptides were evaluated in triple native breast cancer MDA-MB-231 cells and their bone-seeking and lung-seeking sublines, showing reduced cell viability along with significant increased autophagy following treatment in both 2D and 3D cell cultures. Among these new inhibitors, the γ-dipeptide 10b displayed strongest cytotoxicity. The orthotopic NSC mouse model for breast cancer further showed the γ-dipeptides 10b exhibited a superior anticancer effects by inducing autophagic cell death in breast tumor, with no significant systemic toxicity. These findings reveal that Arf1-targeting γ-dipeptides developed by our collaborative team may represent a promising targeted therapeutic to improve treatment of breast cancer. Citation Format: Leilei He, Alain Chavanieu, Yen Vo-Hoang, Yong Teng. Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death [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 1229.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

Abstract PR-005: Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment

Abstract Introduction/Purpose: Radiation therapy drives pro-inflammatory changes to the tumor microenvironment (TME). We have shown that this may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation therapy induces immunosuppressive myeloid cells in the TME in an adenosine-dependent fashion. Methods: Using the 4T1 orthotopic breast cancer mouse model, we tested the immunophenotypic changes to the TME by spectral flow cytometry and bulk RNASeq of tumor infiltrating leukocytes (CD45+, TIL) 72-hours after tumor-directed radiation therapy (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). We cultured human peripheral blood mononuclear cells (PBMCs) with 0.1, 1, and 10uM of adenosine receptor agonist, 5’-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform. Results: Radiation increases the abundance of CD45+CD11b+ myeloid cells (77.4% vs. 84.4%) and decreased CD45+TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME, compared to untreated controls. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a significant reduction Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by an induction of granulocytic and monocytic myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). Radiation caused a decrease in the tumor-associated macrophage population (TAM: 50% vs 31.1%) in the setting of a phenotypic shift to from pro-inflammatory M1-like to immunosuppressive M2-like TAM phenotype: M2-like TAM/M1-like TAM ratio 2.0 vs 13.3. Radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92) by TILs, but not CD73 that metabolizes AMP to adenosine. The addition of NECA to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) at all NECA concentrations tested and a dose-dependent decrease in GM-CSF, IL-12p40 and IL-23. There was a significant reduction of IL-1 β, IL-8, IL-12p70 and MCP-1 only at the highest dose (10uM) tested. IL-6 was increased at the intermediate dose (1uM) of NECA with an overall reduction with high-dose NECA. High-dose NECA alone significantly increased both IFN- β and TGF- β. Conclusion: Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs, M-MDSCs and a shifting the TAM population towards an immunosuppressive phenotype. Radiation increases the expression of adenosine receptors on TILs and treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu suggesting a mechanistic link between radiation and adenosine signaling. Citation Format: Shruti Bansal, Mathew Chaimowitz, Casey R. Ager, Aleksandar A. Obradovic, Charles G. Drake, Catherine S. Spina. Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-005.

Abstract PO-052: Radiation regimen and timing of PD1 blockade critically influence response to combination therapy in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) constitute the sixth most common cancer worldwide, with a 5-year survival of less than 50% for advanced HNSCC patients. Radiation therapy (RT) is widely used in the treatment of HNSCC patients. Preclinical studies have shown that RT can trigger immune mediated response that potentiates tumor regression. However, the use of RT in combination with immune checkpoint inhibitor, specifically programmed death protein-1 blockade (αPD-1), has not been extensively examined in HNSCC. In vitro studies in our laboratory revealed that radiation increased PD-L1 expression in HNSCC tumor cells in a dose-dependent manner. Based on this observation, we hypothesized that the efficacy of RT and αPD-1 therapy would be critically influenced by the timing of αPD-1 of administration. To test this hypothesis, we conducted a preclinical study using a novel orthotopic immunocompetent mouse model of HNSCC (RPMOC1). Tumor bearing mice were randomized into the following arms: RT (single dose 10 Gy), fractionated RT (fRT- 2Gy x 5), RT prior to αPD-1(200 μg), RT post αPD-1, fRT prior to αPD-1 and fRT post αPD-1. Non-invasive magnetic resonance imaging (MRI) was used to monitor tumor response to therapy. All treatment regimens were well tolerated and did not result in weight loss during the experiment. The most potent tumor growth inhibition was observed in the RT (10 Gy) and αPD-1 combination therapy group. RT given either before or after αPD-1 therapy resulted in a significant decrease in tumor volumes (p&amp;lt;0.05) compared to other groups. Combination therapy with fractionated radiation did not show improved response compared to monotherapy. Overall, our observations indicate that tumor response to combined radiation and immunotherapy is critically influenced by the radiation regimen and timing of PD1 blockade in HNSCC. Citation Format: Aparajita Verma, Michael Habitzruther, Kristopher Attwood, Mukund Seshadri. Radiation regimen and timing of PD1 blockade critically influence response to combination therapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-052.

Elevation of miR-302b prevents multiple myeloma cell growth and bone destruction by blocking DKK1 secretion

BackgroundMyeloma bone disease (MBD) is a severe complication of multiple myeloma (MM) mainly due to an imbalance between enhanced osteoclast activity and reduced osteoblast function. Previous studies have demonstrated that miRNAs play a vital role in the osteogenic differentiation of mesenchymal stromal cells (MSCs) in MM. However, the value of miR‑302b in MBD remains to be further elucidated. The aim of this study is to explore the role of miR‑302b in the regulation of MBD osteogenic differentiation and evaluate the potential of a new therapeutic strategy for the clinical treatment of MBD.MethodOur previous research demonstrated that MiR-302b belongs to the miR-302 cluster and is able to inhibit tumor growth and osteolysis in an orthotopic osteosarcoma xenograft tumor mouse model. In this study, we first transfected miR-302b mimics, miR-302b inhibitor, and miR-302b NC into MM1.S and RPMI8226 MM cells to detect the correlation between miR-302b expression in the pathological specimens and the clinicopathological features by qPCR, the target correlation between miR-302b and DKK1 by immunohistochemistry, qPCR and Western blot, and the correlation between miR-302b and the Wnt/β-catenin signaling pathway by Western blot. The effect of miR-302b on osteoblastogenesis was also studied in a subperiosteal tumorigenesis model of NOD/SCID nude mice.ResultsWe found that increased miR-302b suppressed cell proliferation and induced cell apoptosis in RPMI 8226 and MM1.S cells. TargetScan online bioinformatic analysis predicted that miR-302b is able to bind to 3′UTR of DKK1 mRNA. Target binding of miR-302b to DKK1 was demonstrated by dual-luciferase reporter assay, qPCR, Western blot and immunohistochemistry, indicating that miR-302b is able to degrade DKK1 in RPMI 8226 and MM1.S cells. The model of co-culturing MM cells with preosteoblast MC3T3-E1 cells showed that miR-302b inhibits MM-induced suppression of osteoblast differentiation. Western blotting showed that miR-302b promotes the Wnt/β-catenin signaling pathway in MM cells. Micro-CT and immunohistochemistry results showed that miR-302b suppresses myeloma bone destruction in vivo.ConclusionmiR-302b is able to target DKK1 and promote the Wnt/β-catenin signaling pathway in MM.

A multistage oral delivery system of PTX for improving oral bioavailability and enhancing anticancer efficacy

Objective Bromotetrandrine (W198) was reported as a P-glycoprotein (P-gp) inhibitor. We aimed to prepare oral W198 micelles following by paclitaxel (PTX) micelles (W198/PTX micelles) to improve the clinical application of PTX. Significance The poor water solubility, intestinal permeability, and multidrug resistance (MDR) of PTX can be improved in the multistage oral delivery system. Methods The novel W198/PTX oral micelles were developed by water-bath ultrasound method and were evaluated in vivo and in vitro in 4T1 orthotopic tumor-bearing mice model. Results PTX micelles and W198 micelles were prepared to be round and uniform. W198 micelles pre-administrated group showed higher cellular uptake efficiency of PTX on Caco-2 cells and more prominent cytotoxicity compared with W198-untreated group on 4T1 cells. The oral bioavailability of W198/PTX micelles group was nearly 5.7-folds higher than the PTX micelles only group. In addition, W198/PTX micelles showed enhanced anticancer efficacy. Conclusions We established a multistage oral delivery system to improve oral bioavailability and anticancer efficacy of PTX.

MicroRNA-362 negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling to suppress cell migration and invasion.

Cell migration and invasion are modulated by epithelial-to-mesenchymal transition (EMT) and the reverse MET process. Despite the detection of microRNA-362 (miR-362, both the miR-362-5p and -3p species) in cancers, none of the identified miR-362 targets is a mesenchymal or epithelial factor to link miR-362 with EMT/MET and metastasis. Focusing on the TGF-β/SMAD signaling pathway in this work, luciferase assays and western blot data showed that miR-362 targeted and negatively regulated expression of SMAD4 and E-cadherin, but not SNAI1, which is regulated by SMAD4. However, miR-362 knockdown also down-regulated SMAD4 and SNAI1, but up-regulated E-cadherin expression. Wound-healing and transwell assays further showed that miR-362 knockdown suppressed cell migration and invasion, effects which were reversed by over-expressing SMAD4 or SNAI1, or by knocking down E-cadherin in the miR-362 knockdown cells. In orthotopic mice, miR-362 knockdown inhibited metastasis, and displayed the same SMAD4 and E-cadherin expression profiles in the tumors as in the in vitro studies. A scheme is proposed to integrate miR-362 negative regulation via SMAD4, and to explain miR-362 positive regulation of SMAD4 via miR-362 targeting of known SMAD4 suppressors, BRK and DACH1, which would have resulted in SMAD4 depletion and annulment of subsequent involvement in TGF-β signaling actions. Hence, miR-362 both negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling pathway to suppress cell motility and invasiveness and metastasis, and may explain the reported clinical association of anti-miR-362 with suppressed metastasis in various cancers. MiR-362 knockdown in miR-362-positive cancer cells may be used as a therapeutic strategy to suppress metastasis.

Transbronchial fluorescence detection of a porphysome-laden tumor in an orthotopic lung cancer mouse model (mouse 1 in Figure 2). The tumor was localized in the cranial lobe. A part of the tumor at the entrance of the cranial lobe bronchus is easily seen by the composite optical fiberscope (COF), with a sharp contrast in fluorescence relative to the adjacent bronchial

Transbronchial fluorescence detection of a porphysome-laden tumor in an orthotopic lung cancer mouse model (mouse 1 in Figure 2). The tumor was localized in the cranial lobe. A part of the tumor at the entrance of the cranial lobe bronchus is easily seen by the composite optical fiberscope (COF), with a sharp contrast in fluorescence relative to the adjacent bronchial