Flank Tumors Research Articles

Abstract 905: Targeting muscarinic acetylcholine receptors in glioma stem like cells

Abstract Primary gliomas arising within the brain remain the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioblastoma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass that are likely responsible for tumor initiation and recurrence of tumors post resection and give rise to drug resistant tumors. Determining a way to suppress these malignant characteristics and/or depleting the GSC population could improve current cancer treatments and the survival of glioma patients. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), found during neural development and also residing in the adult brain. OPCs are prone to malignant transformation and believed to be a cellular origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We investigated the functional characterization of ACh and mAChRs in modulating malignancy in OPC-like cells. We studied cultures of primary mouse OPC-like GSCs. We used the primary cells to culture three-dimensional tumor organoids in vitro to better represent tumor heterogeneity. We also used patient derived xenografts (PDX) of glioma to grow flank xenografts in NSG mice for an in vivo model. Publicly available data and studies in our lab show high levels of expression of CHRM3 (M3mAChR) in glioma patients and in primary OPC like GSCs. A drug screen conducted in the context of multiple sclerosis determined that benztropine (BZT), an anti muscarinic drug targeting M3mAChR, causes normal OPCs to exit the cell cycle, lose stem cell like characteristics, and differentiate. In our lab, electrophysiological studies demonstrated that activation of mAChRs in OPC-like cells from mouse and patient tumors generates rapid (< 1 second) increases in cytosolic calcium. Pharmacologic studies indicated that treatment with FDA approved anti muscarinic benztropine suppresses proliferation in cultured glioma cells. We also observed that serially passaging flank tumors treated with BZT into new host mice slowed down recurrence of new tumors. In order to determine key receptors mediating cholinergic responses in OPC-like cells we performed protein quantification, which displayed lowered phosphorylation of ERK. Electrophysiological studies are being conducted to dissect the mechanisms by which ACh evokes calcium release. These studies improve understanding of how cholinergic microenvironment influences stem-like glioma cells, providing a platform for repositioning available small molecule modifiers for treatment of glioma. Citation Format: Sumyuktha V. Anand, Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, Matthew C. Havrda. Targeting muscarinic acetylcholine receptors in glioma stem like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 905.

Abstract 293: Identification of a predictive biomarker for liposomal nanoparticle delivery through pan-cancer pooled screening

Abstract Background: Nanoparticles are a modular technology with great promise for cancer treatment, but one obstacle for clinical implementation is a lack of predictive biomarkers to identify patient groups most likely to benefit from specific nanoformulations. We developed a massively parallel pooled screen to investigate genomic factors underlying NP-cancer cell engagement, and report here the identification of a predictive biomarker that is both formulation-specific and cancer lineage agnostic. Methods: We interrogated the interactions of 35 fluorescent nanoparticle formulations (15 liposomal and 25 polymeric) against 488 pooled, barcoded cancer cell lines using fluorescence-activated cell sorting and binned cells based on strength of association. After sequencing, the relative barcode abundance in each bin was used to generate an association score for each nanoparticle-cell line pair. We identified features predictive of NP-cancer cell association by interfacing this score with multi-omic data from the Cancer Cell Line Encyclopedia. Results: Using machine learning model predictions, we identified thousands of candidate biomarkers both across and within formulations. Expression of the lysosomal transporter SLC46A3 was the top ranked random forest feature for all liposome formulations and was prioritized for further study. Univariate analyses confirmed the significance of this candidate biomarker and indicated a negative relationship, such that low expression of SLC46A3 is highly correlated with high nanoparticle association. This inverse relationship held true across all 22 cancer lineages screened and was recapitulated in a non-pooled screen of 13 cancer cell lines with a range of native SLC46A3 expression. To determine whether modulating SLC46A3 expression is sufficient to negatively regulate uptake of liposomes, we developed a toolkit of engineered cell lines by knocking out SLC46A3 in high-expressing T47D cells and inducing overexpression in low-expressing LOXIMVI cells. Consistent with our hypothesis, we show that SLC46A3 expression is inversely correlated with uptake of liposomal, but not polymeric, nanoparticles. To evaluate the potential clinical utility of SLC46A3 as a biomarker, we tested in vivo delivery of an FDA-approved nanoparticle analog, the drug-free version of liposomal irinotecan, to LOXIMVI flank tumors via intratumoral injection and intravenous administration. For both administration methods, we show that low tumor expression of SLC46A3 correlates with significantly improved delivery of liposomal nanoparticles. Conclusions: We identified SLC46A3 as a negative regulator of liposomal nanoparticle delivery to cancer cells both in vitro and in vivo. Given that liposomal nanoparticles comprise the majority of FDA approvals for cancer indications, we propose SLC46A3 may be a clinically actionable biomarker for patient stratification. Citation Format: Joelle P. Straehla, Natalie Boehnke, Hannah Safford, Mustafa Kocak, Matthew G. Rees, Melissa Ronan, Danny Rosenberg, Charles H. Adelmann, Raghu R. Chivukula, Jaime H. Cheah, Hojun Li, Jennifer A. Roth, Angela N. Koehler, Paula T. Hammond. Identification of a predictive biomarker for liposomal nanoparticle delivery through pan-cancer pooled screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 293.

Abstract 2016: Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models

Abstract Introduction: Antibody-drug Conjugates (ADCs) targeting differentially expressed tumor cell-surface antigens show robust clinical activity in several solid tumor cancers, but none is yet available for osteosarcoma. Due to the high unmet medical need in this indication, there is an urgent requirement to identify respective osteosarcoma cell-surface antigens and evaluate the antitumor activity of an ADC that can deliver tailored cytotoxic payloads to osteosarcoma tumors expressing these targets. Methods: We used an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. uPARAP (MRC2) was found to be enriched in osteosarcoma at both protein and mRNA levels. The cell-surface expression of uPARAP (MRC2) was further validated by IHC and flow cytometry with osteosarcoma cell lines, PDX models, and patient tumor tissue microarray. As a proof of concept, three MRC2 targeted ADCs with different Microtubulin- and alkylating agent types of payloads were tested in 8 osteosarcoma PDX models. uPARAP (MRC2) ADC, control ADC, or vehicle control was administered IV to mice harboring flank tumors at a dose of 3mg/kg, weekly X 3 (ADCE-003), 2mg/kg on Day 1 (ADCE-010), and 10mg/kg weekly X 2 (ADCE-011). EFS for treatment (T) and control (C) groups, minimum relative tumor volume (minRTV), and objective response measures were analyzed. Results: Western blotting confirmed the expression of uPARAP (MRC2) in 8 osteosarcoma cell lines and 8 PDX models. Flow cytometry further validated cell-surface localization and expression levels of uPARAP (MRC2) in 7 osteosarcoma cell lines. We then performed immunohistochemistry (IHC) staining using an osteosarcoma tissue microarray from 37 patients and 19 PDX models. uPARAP (MRC2) was expressed in 97% of the patient samples and 95% of the PDXs. 51% of the patient samples and 39% of the PDXs had an overall H-score of 100 or higher. Mice tolerated ADCs well with minimal toxicity. All 3 ADCs significantly prolonged EFS in 6/8 osteosarcoma models. Complete response (CR) or maintained CR were observed in 2 models in ADCE-010 and ADCE-011 groups, respectively. Conclusions: uPARAP (MRC2) is highly expressed in most osteosarcoma samples, which makes it a viable target for respective antigen targeting ADC therapies. Three uPARAP (MRC2) targeting ADCs showed antitumor activities in osteosarcoma preclinical models which warrant further investigation of uPARAP targeting ADC therapies for osteosarcoma. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana Lazcano, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Niels Behrendt, Christoffer F. Nielsen, Lars H. Engelholm, Richard Gorlick. Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2016.

Trastuzumab Deruxtecan, Antibody-Drug Conjugate Targeting HER2, Is Effective in Pediatric Malignancies: A Report by the Pediatric Preclinical Testing Consortium.

HER2 is expressed in many pediatric solid tumors and is a target for innovative immune therapies including CAR-T cells and antibody-drug conjugates (ADC). We evaluated the preclinical efficacy of trastuzumab deruxtecan (T-DXd, DS-8201a), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient- and cell line-derived xenograft (PDX/CDX) models. HER2 mRNA expression was determined using RNA-seq and protein expression via IHC across multiple pediatric tumor PDX models. Osteosarcoma (OS), malignant rhabdoid tumor (MRT), and Wilms tumor (WT) models with varying HER2 expression were tested using 10 mice per group. Additional histologies such as Ewing sarcoma (EWS), rhabdomyosarcoma (RMS), neuroblastoma (NB), and brain tumors were evaluated using single mouse testing (SMT) experiments. T-DXd or vehicle control was administered intravenously to mice harboring established flank tumors at a dose of 5 mg/kg on day 1. Event-free survival (EFS) and objective response were compared between treatment and control groups. HER2 mRNA expression was observed across histologies, with the highest expression in WT (median = 22 FPKM), followed by MRT, OS, and EWS. The relationship between HER2 protein and mRNA expression was inconsistent. T-DXd significantly prolonged EFS in 6/7 OS, 2/2 MRT, and 3/3 WT PDX models. Complete response (CR) or maintained CR (MCR) were observed for 4/5 WT and MRT models, whereas stable disease was the best response among OS models. SMT experiments also demonstrated activity across multiple solid tumors. Clinical trials assessing the efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered.

Lung Inflammation Predictors in Combined Immune Checkpoint-Inhibitor and Radiation Therapy-Proof-of-Concept Animal Study.

Purpose: Combined radiotherapy (RT) and immune checkpoint-inhibitor (ICI) therapy can act synergistically to enhance tumor response beyond what either treatment can achieve alone. Alongside the revolutionary impact of ICIs on cancer therapy, life-threatening potential side effects, such as checkpoint-inhibitor-induced (CIP) pneumonitis, remain underreported and unpredictable. In this preclinical study, we hypothesized that routinely collected data such as imaging, blood counts, and blood cytokine levels can be utilized to build a model that predicts lung inflammation associated with combined RT/ICI therapy. Materials and Methods: This proof-of-concept investigational work was performed on Lewis lung carcinoma in a syngeneic murine model. Nineteen mice were used, four as untreated controls and the rest subjected to RT/ICI therapy. Tumors were implanted subcutaneously in both flanks and upon reaching volumes of ~200 mm3 the animals were imaged with both CT and MRI and blood was collected. Quantitative radiomics features were extracted from imaging of both lungs. The animals then received RT to the right flank tumor only with a regimen of three 8 Gy fractions (one fraction per day over 3 days) with PD-1 inhibitor administration delivered intraperitoneally after each daily RT fraction. Tumor volume evolution was followed until tumors reached the maximum size allowed by the Institutional Animal Care and Use Committee (IACUC). The animals were sacrificed, and lung tissues harvested for immunohistochemistry evaluation. Tissue biomarkers of lung inflammation (CD45) were tallied, and binary logistic regression analyses were performed to create models predictive of lung inflammation, incorporating pretreatment CT/MRI radiomics, blood counts, and blood cytokines. Results: The treated animal cohort was dichotomized by the median value of CD45 infiltration in the lungs. Four pretreatment radiomics features (3 CT features and 1 MRI feature) together with pre-treatment neutrophil-to-lymphocyte (NLR) ratio and pre-treatment granulocyte-macrophage colony-stimulating factor (GM-CSF) level correlated with dichotomized CD45 infiltration. Predictive models were created by combining radiomics with NLR and GM-CSF. Receiver operating characteristic (ROC) analyses of two-fold internal cross-validation indicated that the predictive model incorporating MR radiomics had an average area under the curve (AUC) of 0.834, while the model incorporating CT radiomics had an AUC of 0.787. Conclusions: Model building using quantitative imaging data, blood counts, and blood cytokines resulted in lung inflammation prediction models justifying the study hypothesis. The models yielded very-good-to-excellent AUCs of more than 0.78 on internal cross-validation analyses.

LDH-A-Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses.

Simple SummaryThree different murine glioma cell lines were modified to downregulate the expression of the murine LDH-A gene using shRNA knock-down (KD), and compared to shRNA scrambled control (NC) cell lines. Successful LDH-A KD was confirmed by differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles. mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines (CT2A, and ALTS1C1) do not have this capacity. The data suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment. Furthermore, LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors. Our results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed with a capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion, leading to a difference in growth and survival in an immune-competent environment.Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.

Specificity of Binding and Antitumor Efficacy of a Covalent Microtubule Stabilizer

Microtubule stabilizers, including the taxanes paclitaxel and docetaxel, are a mainstay in the treatment of solid tumors, including breast and ovarian cancers. Although these agents are often effective as first‐line therapy, they are susceptible to common drug resistance mechanisms, including the upregulation of drug efflux pumps. Therefore, there is a critical need to develop novel classes of microtubule targeted agents that retain activity in taxane‐resistant settings. The C22,23‐epoxy taccalonolides are a class of plant‐derived microtubule stabilizers that covalently and irreversibly bind microtubules, allowing them to retain efficacy in a variety of taxane‐resistant cancer models. We employed cellular‐based binding studies to fully map the taccalonolide pharmacophore and confirm the target specificity of this covalent compound. The identification of β‐tubulin residues that are critical for taccalonolide binding in a cellular context has provided the opportunity to generate functional taccalonolide mimetics through a synthetically tractable approach.Herein, we demonstrate that taccalonolide AF is less susceptible to p‐glycoprotein‐mediated drug efflux than paclitaxel in two separate ovarian cancer models and that it has superior cellular persistence after drug washout, likely due to irreversible target engagement. While taccalonolide AF demonstrated potent and persistent in vivo antitumor efficacy against the taxane‐resistant NCI/ADR‐RES ovarian cancer model as a flank xenograft when administered systemically, it was associated with a narrow therapeutic window. Additional studies showing that intratumoral injection of the taccalonolides into flank tumors provided long‐term antitumor efficacy months after the final dose prompted a study of their utility in the targeted delivery to a disseminated orthotopic ovarian cancer model representing localized metastasis. Indeed, we found that taccalonolide‐treated animals showed a significant decrease in micrometastasis of taxane‐resistant ovarian cancer cells to the spleen, as detected by quantitative RT‐PCR, without any evidence of systemic toxicity. We propose that the irreversible mechanism of microtubule stabilization elicited by the taccalonolides has the unique potential for intraperitoneal treatment of locally disseminated taxane‐resistant disease, which represents a significant unmet clinical need in the treatment of ovarian cancer patients.

PD53-11 DIVERGENT THERAPEUTIC OUTCOMES OF STING AGONIST ADU-S100 IN INTRATUMORAL AND INTRAVESICAL TREATMENT REGIMENS IN SYNGENEIC MURINE MB49 AND IN THE N-METHYL-N-NITROSOUREA (MNU) RAT MODEL OF UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD53-11 DIVERGENT THERAPEUTIC OUTCOMES OF STING AGONIST ADU-S100 IN INTRATUMORAL AND INTRAVESICAL TREATMENT REGIMENS IN SYNGENEIC MURINE MB49 AND IN THE N-METHYL-N-NITROSOUREA (MNU) RAT MODEL OF UROTHELIAL CARCINOMA Alok Kumar Singh, Kara Lombardo, Monali Praharaj, James Liu, Russel Becker, Kelly Harris, Max Kates, David McConkey, Andres Matoso, William R. Bishai, and Trinity Jude Bivalacqua Alok Kumar SinghAlok Kumar Singh More articles by this author , Kara LombardoKara Lombardo More articles by this author , Monali PraharajMonali Praharaj More articles by this author , James LiuJames Liu More articles by this author , Russel BeckerRussel Becker More articles by this author , Kelly HarrisKelly Harris More articles by this author , Max KatesMax Kates More articles by this author , David McConkeyDavid McConkey More articles by this author , Andres MatosoAndres Matoso More articles by this author , William R. BishaiWilliam R. Bishai More articles by this author , and Trinity Jude BivalacquaTrinity Jude Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: STING agonist ADU-S100 as an intratumoral (IT) therapeutic regimen shows excellent CD8+T cells-mediated antitumor immunity. Rapid absorption from IT sites, short terminal half-life and treatment related adverse outcomes caused withdrawal of ADU-S100 from clinical trials. We developed BCG-STING, a preclinical candidate for non-muscle invasive bladder cancer (NMIBC) that overexpresses a STING agonist (c-di-AMP). BCG-STING confers superior antitumor immunity over BCG-WT by increasing tumor infiltrating CD4+ and CD8+ T cells and inflammatory macrophages. Due to similarities in the mechanism of action, we compared antitumor efficacy of BCG-STING with ADU-S100 in an IT as well as intravesical (IV) dosing regimen. METHODS: Syngeneic MB49 flank tumors in C57BL/6 female mice were given IT treatment of BCG-WT, BCG-STING (5x106CFU) or ADU-S100 (100, 50 or 25 mg). End-point measurements included tumor volume and flow-cytometry based tumor immune infiltrate analyses (at 100 μg). MNU carcinogen rat model of NMIBC and the standard IV administration regimen was used for BCG-WT or BCG-STING (5x106 CFU, 6x weekly) or ADU-S100 (25 μg, 6x weekly). Histopathological analyses of MNU rat bladders were performed for tumor involvement index (TII) and pathological tumor staging. RESULTS: IT administration of ADU-S100 in MB49 tumor showed greatest tumor regression over BCG-WT or BCG-STING even at lowest dose (25 μg). ADU-S100 caused strongest infiltration of TNF-α+MHCII+F4/80+CD11b+ macrophages and IFN-γ+CD8+ T cells as compared to BCG-STING or BCG-WT. We did observe a significant increase in immunosuppressive IL-10+ and ARG-1+ Ly6C(hi)Ly6G(-) monocytic myeloid-derived suppressor cells (M-MDSCs) in MB49 tumors treated with ADU-S100 and BCG-WT, but not BCG-STING. In contrast to MB49 model, lV induction course of BCG-STING in MNU rat model showed the greatest antitumor effects with only 5% residual TII compared to 30% in ADU-S100 or 42% in BCG-WT. Tumor staging revealed residual T1 (50%), CIS (25%) and Ta (25%) tumors in ADU-S100 group, while BCG-WT treated group showed a lower degree of invasion to the lamina propria with CIS (50%), T1 (25%) and Ta (25%) residual tumors. BCG-STING IV therapy resulted in 60% of rat bladders showing complete tumor regression while 40% had minimal residual non-invasive tumors. CONCLUSIONS: The divergent therapeutic outcomes of IT vs IV treatment regimens of ADU-S100 over BCG-STING or BCG-WT in different urothelial carcinoma models indicate the critical role of tumor microenvironment and dosing regimens on relative efficacy. Induction of immunosuppressive M-MDSCs by ADU-S100 or BCG-WT suggests unique advantages of BCG-STING. The therapeutic targeting of M-MDSCs as combination may improve therapeutic efficacies of BCGs. Source of Funding: National Institute of Health, Maryland Tedco, Willowcroft Foundation, and Cigarette Restitution Fund © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e914 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alok Kumar Singh More articles by this author Kara Lombardo More articles by this author Monali Praharaj More articles by this author James Liu More articles by this author Russel Becker More articles by this author Kelly Harris More articles by this author Max Kates More articles by this author David McConkey More articles by this author Andres Matoso More articles by this author William R. Bishai More articles by this author Trinity Jude Bivalacqua More articles by this author Expand All Advertisement PDF downloadLoading ...

Enhanced anti-tumor immunity in ST8Sia6 knockout mice

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6 on O-linked glycoproteins and functions to inhibit activation of innate immune cells. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity. Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery

Testosterone functions as a tumor suppressor in glioblastoma

Abstract A sex difference in the incidence and outcome of patients with glioblastoma (GBM), the most common primary malignant brain tumor, has been well established, as male patients experience a worse prognosis. However, the contribution of sex hormones to anti-tumor immunity has not been fully explored. To test this, we orthotopically implanted syngeneic mouse GBM cells into male mice after castration or sham surgery to assess survival differences. Castrated immune-competent mice succumbed to tumors significantly faster than the sham surgery group, and this was rescued by exogenous testosterone administration. In contrast, immune-deficient NSG castrated mice survived longer than sham animals, suggesting a role for immune cells in the survival difference. Flow cytometry analysis of tumor-infiltrating immune cells further showed decreased cytokine production in T cells from castrated mice, while no difference was found in immune cell infiltration. Flank tumor experiments suggested brain-specific regulation of T cell recruitment, as tumor growth was delayed in castrated mice, with higher infiltration of CD8+ T cells but decreased cytokine production. Surprisingly, mass spectrometry of serum revealed upregulated levels of circulating corticosterone in castrated mice regardless of tumor presence, suggesting that castration may induce systemic immunosuppression via upregulation of glucocorticoids. Taken together, our data demonstrate that testosterone regulates T cell immunity, possibly via glucocorticoids, and ultimately affects GBM tumor growth. Given the decreased level of testosterone in aged males with higher risk of GBM, these findings further suggest the potential therapeutic intervention of testosterone in GBM. Supported by grants from NIH (P01 CA245705)

Pre-Clinical Study Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Neuroblastoma.

Simple SummaryDespite significant advancements in neuroblastoma therapy, over half of the children with high-risk neuroblastoma will suffer disease relapse. Protein phosphatase 2A (PP2A) is a tumor suppressor that is decreased in neuroblastoma. Previous studies with FTY720, an immunomodulatory agent that activates PP2A, led to a decreased malignant phenotype in neuroblastoma. To activate PP2A without issues associated with the use of an immunomodulator, we investigated two novel PP2A-activating compounds, ATUX-792 and DBK-1154, designed for improved efficacy and fewer side effects. We aimed to demonstrate that ATUX-792 and DBK-1154 would decrease neuroblastoma cell survival and tumor growth with the goal of providing pre-clinical data to support advancing these compounds into the clinical arena.Background: Protein phosphatase 2A (PP2A) functions as an inhibitor of cancer cell proliferation, and its tumor suppressor function is attenuated in many cancers. Previous studies utilized FTY720, an immunomodulating compound known to activate PP2A, and demonstrated a decrease in the malignant phenotype in neuroblastoma. We wished to investigate the effects of two novel PP2A activators, ATUX-792 (792) and DBK-1154 (1154). Methods: Long-term passage neuroblastoma cell lines and human neuroblastoma patient-derived xenograft (PDX) cells were used. Cells were treated with 792 or 1154, and viability, proliferation, and motility were examined. The effect on tumor growth was investigated using a murine flank tumor model. Results: Treatment with 792 or 1154 resulted in PP2A activation, decreased cell survival, proliferation, and motility in neuroblastoma cells. Immunoblotting revealed a decrease in MYCN protein expression with increasing concentrations of 792 and 1154. Treatment with 792 led to tumor necrosis and decreased tumor growth in vivo. Conclusions: PP2A activation with 792 or 1154 decreased survival, proliferation, and motility of neuroblastoma in vitro and tumor growth in vivo. Both compounds resulted in decreased expression of the oncogenic protein MYCN. These findings indicate a potential therapeutic role for these novel PP2A activators in neuroblastoma.

Radiation dose and schedule influence the abscopal effect in a bilateral murine CT26 tumor model

Radiotherapy (RT) can induce immune-mediated responses in local irradiated tumors, and non-irradiated distant metastasis is termed the abscopal effect. Here, we aimed to evaluate the impact of different RT doses and fractions on anti-tumor responses within local irradiated and distance non-irradiated tumor microenvironments. In mice bearing CT26 tumors, the primary tumor was irradiated with three different RT doses (16 Gy × 1F, 10 Gy × 2F, and 3 Gy × 10F) with the same biologically effective dose. Tumor volumes and immune cells changes were assessed in irradiated and non-irradiated tumors. Survival times were evaluated over 90 days. Only 16 Gy × 1F radiation increased CD8 + T cells number in the irradiated (p = 0.043) and non-irradiated (p = 0.047) tumors compared to the untreated group. A high frequency of tumor-associated macrophages-1 (TAM-1) and low TAM-2 was found in 16 Gy × 1F irradiated mice.Moreover, 16 Gy × 1F significantly induced interferon gamma (IFNγ)-producing CD8 + cells in the spleen compared to controls (p = 0.021). Hypofraction regimens (16 Gy × 1F, 10 Gy × 2F) caused a reduction in myeloid-derived suppressor cells in the irradiated tumors. We detected A modest growth delay in both flank tumors and long-term survival after hypofraction treatments (16 Gy × 1F, 10 Gy × 2F). A single high RT dose increased CD8 + cells number in irradiated (p = 0.000) and non-irradiated (p = 0.002) tumors approximal up to 2 points along with significant induction of IFN-γ production by CD8 + cells in the spleen when combined with anti- programmed death ligand-1 (PDL-1) (p = 0.000). Combination therapy was also associated with bilateral tumor growth control and increased life span in mice. Hypofractionated RT schedules, especially single high dose, seem the most effective regimen for inducing an abscopal effect. Immune checkpoint inhibitors could promote RT-induced systemic effects.

Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways.

Simple SummaryThe oncogene MYC alters cellular metabolism. Medulloblastoma is the most common malignant pediatric brain tumor. MYC-amplified medulloblastoma has a poor prognosis, and the metabolism of MYC-amplified medulloblastoma is poorly understood. We performed comprehensive metabolic profiling of MYC-amplified medulloblastoma and found increased reliance on potentially targetable pathways. We also found that the metabolism of MYC-amplified cell lines differed from orthotopic brain tumors in vitro and in flank tumors, suggesting that analyses conducted in vitro or in flank tumors may miss key vulnerabilities.Reprograming of cellular metabolism is a hallmark of cancer. Altering metabolism allows cancer cells to overcome unfavorable microenvironment conditions and to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC defines a subset of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in three different conditions—in vitro, in flank xenografts and in orthotopic xenografts in the cerebellum. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and in vitro MYC-amplified cells. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of the TCA cycle as well as the synthesis of nucleotides, hexosamines, amino acids and glutathione. There was significantly higher glucose uptake and usage in orthotopic xenograft tumors compared to flank xenograft tumors and cells in culture. In orthotopic tumors, glucose was the main carbon source for the de novo synthesis of glutamate, glutamine and glutathione through the TCA cycle. In vivo, the glutaminase II pathway was the main pathway utilizing glutamine. Glutathione was the most abundant upregulated metabolite in orthotopic tumors compared to normal brain. Glutamine-derived glutathione was synthesized through the glutamine transaminase K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have different metabolic profiles in vitro compared to in vivo, and key vulnerabilities may be missed by not performing in vivo metabolic analyses.

Abstract P011: B cell subsets that correlate with anti-PD-1 resistance in a preclinical model of HPV+ oropharyngeal cancer

Abstract A subset of head and neck squamous cell carcinomas (HNSCC) is associated with infection with oncogenic strains of human papillomavirus (HPV) and its prevalence continues to rise in the US. Cancer progression has been associated, in part, with an impaired immunity. Because of this, immunotherapies such as immune checkpoint blockade (ICB) have been FDA approved to treat different types of cancers, including HNSCC. The ICB targeting the molecule programmed death cell-1 (PD-1) is used as treatment for HPV-related HNSCC, however, only around 30% of patients respond. Most studies have focused on characterizing T cell responses, however, B cells, are also present in the tumor microenvironment of HPV-positive HNSCC. Interestingly, different B cell subsets with differential functions that may promote or prevent an effective anti-tumor response are present in tumors and tumor-draining lymph nodes. Therefore, fine regulation of anti-tumor and tumor-promoting B cell subsets may be necessary to promote an effective anti-tumor response and therefore prevent tumor progression. However, the B cells that may be associated with responsiveness to anti-PD-1 in HPV+ oropharyngeal cancer remain undefined. By using the preclinical model for HPV+ oropharyngeal cancer (named mEER) we have determined that tongue-implanted tumors are sensitive to anti-PD-1, where around 50% of the mice clear the tumors, whereas flank-implanted tumors are completely resistant to these treatments. By using this in vivo model, we were able to characterize B cell subsets in anti-PD-1 sensitive and anti-PD-1 resistant tumors. First, we compared B cell subsets in untreated mice between tongue (anti-PD-1 sensitive) and flank-implanted (anti-PD-1 resistant) tumors by flow cytometry. Interestingly, flank-implanted tumors contained more PD-1-expressing B cells, which have been identified as an immunosuppressive population in hepatocellular carcinoma. The presence of immunosuppressive B cells on flank tumors was also supported by our finding that flank implanted tumors grew at a slower rate in B cell-deficient mice compared to wild-type mice. When we compared tongue-bearing tumor mice that responded or not responded to anti-PD-1, we observed that mice that did not respond to anti-PD-1 had a higher frequency of antibody-secreting cells in tumor-draining lymph nodes compared to responders. Lastly, when we cultured naïve B cells with tumor cell supernatant from mEER cells, B cells upregulated PD-1 and were able to produce the immunosuppressive cytokine IL-10. Our data suggest that HPV+ oropharyngeal tumors modulate B cell functions and that a differential infiltration of B cell subsets into tumors and tumor-draining lymph nodes correlates with responsiveness to anti-PD-1 therapy in a preclinical model of HPV+ oropharyngeal cancer. These results will aid in our long-term goal to elucidate novel mechanisms for the modulation of anti-tumor responses by B cells and the development of novel B cell-specific therapies for HPV-related cancers and, therefore, improve clinical outcomes in these patients. Citation Format: Pamela A. Merheb, Daniel L. Castañón, Michael Rivera, Jorge Galán, Stephanie M. Dorta-Estremera. B cell subsets that correlate with anti-PD-1 resistance in a preclinical model of HPV+ oropharyngeal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P011.

Preclinical Evaluation of an Activity-Based Probe for Intraoperative Imaging of Esophageal Cancer.

Background Early detection and complete resection are important prognostic factors for esophageal cancer (EC). Intraoperative molecular imaging (IMI) using tumor-targeted tracers is effective in many cancer types. However, there are no EC-specific IMI tracers. We sought to test a cathepsin activity-based tracer (VGT-309) for EC resection. Methods Murine (AKR, HNM007) and human (OE19) EC cell lines were screened for cathepsin expression by western blotting. In vitro binding affinity of VGT-309 was evaluated by fluorescence microscopy. Flank tumor models were developed by injecting EC cells into the flanks of BALB/c or athymic nude mice. Mice pretreated with a cathepsin inhibitor (JPM-OEt) were used to confirm on target binding. Animals were injected with 2 mg/kg VGT-309, underwent IMI, and were sacrificed 24 hours after injection. Results Cathepsins B, L, S, and X were expressed by EC cell lines, and all cell lines were labeled in vitro with VGT-309. Fluorescent signal was eliminated when cells were pretreated with JPM-OEt. On biodistribution analysis, VGT-309 accumulated in the liver, kidneys, and spleen without other organ involvement. VGT-309 selectively accumulated in flank allografts and xenografts, with mean signal-to-background ratio of 5.21 (IQR: 4.18-6.73) for flank allografts and 4.34 (IQR: 3.75-5.02) for flank xenografts. Fluorescence microscopy and histopathological analysis confirmed the selective accumulation of the tracer in tumors compared to background normal tissues. Conclusions VGT-309 is an effective tracer for IMI of esophageal cancer. There is potential for clinical translation both as an adjunct to endoscopic detection and for complete removal of disease during esophagectomy.

Short-course neoadjuvant in situ vaccination for murine melanoma

BackgroundSurgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the...

Cancer-Erythrocyte Hybrid Membrane-Camouflaged Magnetic Nanoparticles with Enhanced Photothermal-Immunotherapy for Ovarian Cancer

Cell-membrane-coated nanoparticles are widely studied due to their inherent cellular properties, such as immune escape and homologous homing. A cell membrane coating can also maintain the relative stability of nanoparticles during circulation in a complex blood environment through cell membrane encapsulation technology. In this study, we fused a murine-derived ID8 ovarian cancer cell membrane with a red blood cell (RBC) membrane to create a hybrid biomimetic coating (IRM), and hybrid IRM camouflaged indocyanine green (ICG)-loaded magnetic nanoparticles (Fe3O4-ICG@IRM) were fabricated for combination therapy of ovarian cancer. Fe3O4-ICG@IRM retained both ID8 and RBC cell membrane proteins and exhibited highly specific self-recognition of ID8 cells in vitro and in vivo as well as a prolonged circulation lifetime in blood. Interestingly, in the bilateral flank tumor model, the IRM-coated nanoparticles also activated specific immunity, which killed homologous ID8 tumor cells but had no effect on B16-F10 tumor cells. Furthermore, Fe3O4-ICG@IRM showed synergistic photothermal therapy, resulting in the release of whole-cell tumor antigens by photothermal-induced tumor necrosis, which further enhanced antitumor immunotherapy for primary tumor and metastatic tumor by activating CD8+ cytotoxic T cells and reducing regulatory Foxp3+ T cells. Together, the biomimetic Fe3O4-ICG@IRM nanoparticles showed synergistic photothermal-immunotherapy for ovarian cancer.

Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models.

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

EXTH-24. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE SECOND-GENERATION HDAC INHIBITOR, QUISINOSTAT

Abstract BACKGROUND Class I Histone deacetylases (HDACs) are highly expressed in glioblastoma (GBM) and are considered promising therapeutic targets for cancer treatment. Quisinostat is a class I HDAC inhibitor with high specificity for HDAC1 and 2. In this study, we evaluated the pharmacokinetics (PK), pharmacodynamics (PD), and radiation sensitization properties of quisinostat in orthotopic and flank human glioma models of GBM. METHODS In vitro drug-uptake and PK-PD correlation experiments were conducted in patient-derived glioma stem cell (GSC) lines. Pilot PK studies were performed in mice to optimize the dosing regimen and administration route (subcutaneous (s.q.), intraperitoneal (i.p.) and oral gavage). Athymic mice with intracranial or flank tumors were treated with the most optimized treatment schedule to evaluate plasma PK profile and brain/tumor distribution of quisinostat. The drug levels in plasma, normal brain, and tumor-tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Western blot analysis and immunocytochemistry were performed to assess changes in acetylated histone, Ki67 (proliferation) and cleaved caspase 3 (cell death) levels. Tumor-bearing mice were randomized to four cohorts – vehicle, radiotherapy, quisinostat monotherapy, and quisinostat plus radiotherapy. RESULTS Quisinostat exhibited potent growth inhibition in multiple GSC lines (IC50 ~60 nM), radiosensitization, increased histone acetylation, and decreased expression of key stem cell markers. Significant toxicity was observed at 10 mg/kg s.q. dosing while the same dosing via i.p. route was tolerated relatively well. The unbound concentration of quisinostat was 19 nmol/L, 0.03 nmol/kg and 0.03 nmol/kg in plasma, tumor, and brain tissue, respectively, at 2 hours post-dose. Pharmacodynamic changes were observed in flank but not in intracranial tumors. CONCLUSION Our data indicate low free quisinostat levels in the brain primarily due to drug instability.

CSIG-08. ABT-414 RESISTANT GBM TUMORS HARBOR THE TEK S466I MUTATION AND EXHIBIT INCREASED STEMNESS

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, remains uniformly fatal due to the lack of effective targeted therapies for this aggressive malignancy. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. The incidence of EGFR alterations in GBM makes inhibition of EGFR/EGFRvIII an attractive therapeutic approach. Depatuxizumab mafodotin (ABT-414) is an antibody-drug conjugate comprised of ABT-806, a mAB against EGFR, and a cytotoxic payload (monomethyl auristatin F). ABT-414 therapy initially showed promising results as an EGFRvIII therapy in vivo and in vitro; however, ABT414 failed to provide a survival benefit in phase I/II clinical trials, potentially due to therapeutic resistance. In this study, we seek to investigate the mechanisms of resistance to ABT-414 by performing whole exome, transcriptome and single cell RNA seq on ABT-414 resistant GBM PDX tumors. Our data showed an enrichment of mutations unique to the ABT-414 resistant tumors, including a point mutation (S466I) in TEK/TIE2 transmembrane angiopoietin receptor. In vitro expression of TEK S466I in GBM cells showed an increase in activation of ERK and STAT3 and increased TEK/TIE2 immunoprecipitation with EGFR compared to the wild-type TEK receptor. Furthermore, gene ontology analysis reveals that ABT-414-treated flank tumors exhibit increased activation of extracellular matrix organization and CNS developmental processes compared to flank tumors in the control treatment groups. ABT-414-treated tumors also demonstrate increased expression of inhibitor of differentiation (ID)1 and ID3, associated with stem-like phenotype in glioblastoma. Taken together, our data indicate that resistance to ABT-414 is mediated by both de novo mutations not detected in the parent tumor and adaptive dysregulation of pathways which may lead to dedifferentiation and therapeutic resistance.