Mouse Sarcoma Research Articles

Synthesis of S-alkylated oxadiazole bearing imidazo[2,1-b]thiazole derivatives targeting breast cancer: In vitro cytotoxic evaluation and in vivo radioactive tracing studies

Breast cancer is the most common invasive cancer diagnosed in women, accounting for most cancer-related fatalities globally. Numerous investigations have revealed that breast cancer is characterized by abnormal expression and maintenance of EGFR levels. In terms of structural study and optimization of several EGFR inhibitors, two series of oxadiazole bearing imidazo[2,1-b]thiazole derivatives were designed and synthesized as potential EGFR inhibitors and assessed for their antitumor activity at NCI-USA. Four derivatives 3b, 3c, 3d and 3e elicited remarkable GI% against MDA-MB-468, T-47D and MCF-7 breast cancer cell lines. Thereafter, MTT assay was performed to reveal that compounds 3b (IC50 = 2.27 µM) and 3d (IC50 = 1.46 µM) showed promising cytotoxic activity against MCF-7 and MDA-MB-468 cell lines, respectively, compared to their reference drugs. Compounds 3b, 3d and 3e revealed good selectivity toward tumor cells with reasonable safety profile when tested against the normal cell line MCF-10a. In vitro EGFR inhibitory assay demonstrated that compounds 3b (IC50 = 0.099 µM) and 3d (IC50 = 0.086 µM) exhibited comparable inhibitory activity to the standard drug erlotinib (IC50 = 0.046 µM). A flow cytometric analysis demonstrated that derivatives 3b and 3d arrested the cell cycle at the S phase in MCF-7 and MDB-MB-468, respectively. Furthermore, the most active derivative 3d was subjected to in vivo radioactive studies. In-vivo biodistribution of 99mTc-3d complex showed a notable elevated accumulation in the targeted sarcoma muscle, indicating the targeting capacity of compound 3d in the tumor of sarcoma mice model. The binding mode of compounds 3b and 3d with EGFR was studied by molecular docking and was further inspected by molecular dynamic simulations. Both compounds were shown to be stable during the course of simulation and demonstrated a plausible interaction pattern with the EGFR binding pocket.

Comparison of the accumulation manner of a macromolecular drug between two mouse tumour models: study with magnetic resonance imaging and the model macromolecular drug, gadolinium-conjugated dextran

A knowledge of the difference of spatio-temporal behaviour of nanomedicine in different type of tumour models is important to develop well-targeted nanomedicine for tumour. In this study, intratumoral accumulation of the model nanomedicine, gadolinium-conjugated dextran (Gd-Dex), was examined with magnetic resonance imaging in two tumour models; mouse sarcoma S180 and radiation-induced mouse fibrosarcoma RIF-1. From time-course of the distribution images, the plasma-to-tumour interstitial tissue transfer constant (Ktrans ) and fractional plasma volume (Vp ) were calculated and mapped. Gd-Dex preferentially distributed to the marginal region of S180 tumours immediately after its injection, and then started to accumulate in some parts of the central region. Ktrans and Vp values were large in the marginal region, while only Ktrans was large in some parts of the central region. In contrast, the distribution of Gd-Dex in RIF-1 tumours was fairly homogeneous, and may have resulted from the homogeneous distributions of Ktrans and Vp . The amounts of Gd-Dex that accumulated in entire tumours in both tumour models correlated with the volume of tumours; however, accumulation in large S180 tumours deviated from the correlation in the early phase. The differences in the manner and pharmacokinetics of nanomedicine among tumour models may affect the accumulation of the medicine.

Xenografting Human Musculoskeletal Sarcomas in Mice, Chick Embryo, and Zebrafish: How to Boost Translational Research.

Musculoskeletal sarcomas pose major challenges to researchers and clinicians due to their rarity and heterogeneity. Xenografting human cells or tumor fragments in rodents is a mainstay for the generation of cancer models and for the preclinical trial of novel drugs. Lately, though, technical, intrinsic and ethical concerns together with stricter regulations have significantly curbed the employment of murine patient-derived xenografts (mPDX). In alternatives to murine PDXs, researchers have focused on embryonal systems such as chorioallantoic membrane (CAM) and zebrafish embryos. These systems are time- and cost-effective hosts for tumor fragments and near-patient cells. The CAM of the chick embryo represents a unique vascularized environment to host xenografts with high engraftment rates, allowing for ease of visualization and molecular detection of metastatic cells. Thanks to the transparency of the larvae, zebrafish allow for the tracking of tumor development and metastatization, enabling high-throughput drug screening. This review will focus on xenograft models of musculoskeletal sarcomas to highlight the intrinsic and technically distinctive features of the different hosts, and how they can be exploited to elucidate biological mechanisms beneath the different phases of the tumor's natural history and in drug development. Ultimately, the review suggests the combination of different models as an advantageous approach to boost basic and translational research.

Evaluating [225Ac]Ac-FAPI-46 for the treatment of soft-tissue sarcoma in mice

PurposeFibroblast Activation Protein (FAP) is an emerging theranostic target that is highly expressed on cancer-associated fibroblasts and on certain tumor cells including sarcoma. We investigated the anti-tumor efficacy of [225Ac]Ac-FAPI-46 as monotherapy or in combination with immune checkpoint blockade (ICB) in immunocompetent murine models of sarcoma sensitive or resistant to ICB.Methods[68Ga]Ga- and [225Ac]Ac-FAPI-46 were tested in subcutaneous FAP+ FSA fibrosarcoma bearing C3H/Sed/Kam mice. The efficacy of up to three cycles of 60 kBq [225Ac]Ac-FAPI-46 was evaluated as monotherapy and in combination with an anti-PD-1 antibody. Efficacy of [225Ac]Ac-FAPI-46 and/or ICB was further compared in FAP-overexpressing FSA (FSA-F) tumors that were sensitive to ICB or rendered ICB-resistant by tumor-induction in the presence of Abatacept.Results[225Ac]Ac-FAPI-46 was well tolerated up to 3 × 60 kBq but had minimal effect on FSA tumor growth. The combination of three cycles [225Ac]Ac-FAPI-46 and ICB resulted in growth delay in 55% of mice (6/11) and partial tumor regression in 18% (2/11) of mice. In FSA-F tumors with FAP overexpression, both [225Ac]Ac-FAPI-46 and ICB were effective without additional benefits from the combination. In locally immunosuppressed and ICB resistant FAP-F tumors, however, [225Ac]Ac-FAPI-46 restored responsiveness to ICB, resulting in significant tumor regression and tumor-free survival of 56% of mice in the combination group up to 60 days post treatment.Conclusion[225Ac]Ac-FAPI-46 efficacy is correlated with tumoral FAP expression levels and can restore responsiveness to PD-1 ICB. These data illustrate that careful patient selection based on target expression and rationally designed combination therapies are critically important to maximize the therapeutic impact of FAP-targeting radioligands.

Spotlight on the treatment of non-small cell lung cancer with rare genetic alterations and brain metastasis: Current status and future perspectives.

In patients with non-small cell lung cancer (NSCLC), oncogenic variants present in <5% of cases are considered rare, the predominant of which include human epidermal growth factor receptor 2 (HER2) mutations, mesenchymal-epithelial transition (MET) alterations, c-ros oncogene 1 (ROS1) rearrangements, rearrangement during transfection (RET) fusions, v-raf mouse sarcoma virus oncogene homolog B1 (BRAF) mutations, and neurotrophic troponin receptor kinase (NTRK) fusions. Brain metastases (BMs) occur in approximately 10%-50% of patients with NSCLC harboring rare genetic variants. The recent advent of small-molecule tyrosine kinase inhibitors and macromolecular antibody-drug conjugates (ADCs) has conferred marked survival benefits to patients with NSCLC harboring rare driver alterations. Despite effective brain lesion control for most targeted agents and promising reports of intracranial remission associated with novel ADCs, BM continues to be a major therapeutic challenge. This review discusses the recent advances in the treatment of NSCLC with rare genetic variants and BM, with a particular focus on intracranial efficacy, and explores future perspectives on how best to treat these patients.

Structural characteristics and anti-tumor effect of low molecular weight Dendrobium officinale polysaccharides by reconstructing tumor microenvironment

In this study, glucanase and mannanase were selected to hydrolysis DOP to obtain low molecular weight DOP (LMWDOP), namely glucanase-hydrolysis Dendrobium officinale polysaccharides (GDOP) and mannanase-hydrolysis Dendrobium officinale polysaccharides (MDOP). The antitumor activity of LMWDOPs and its effect on tumor immune microenvironment were investigated. The growth of transplantable S180 sarcoma in mice was significantly inhibited by GDOP, and MDOP in a dose-dependent manner compared with the model group (P < 0.05, P < 0.01). This indicated that mannanase-hydrolysis Dendrobium officinale polysaccharides have stronger anti-tumor activity. The structure of MDOP was systematically characterized to explore the structure–activity relationship. The average molecular weight of MDOP was 3494 Da. MDOP had a linear chain structure consisting mainly of →6)-α-d-Glcp-(1→ and →4)-β-d-Manp-(1 → residues. O-acetyl group linked to C2 of →4)-β-d-Manp-(1 → residue. In conclusion, it has been determined that the small molecule dendrobium polysaccharides obtained from the hydrolysis of mannanase have good anti-tumor activity and can reconstruct the immune microenvironment. “→ 6-α-d-Glcp- (1 → 4)-β-d-Manp-(1→) may be the structural basis for Dendrobium officinale polysaccharides to exert anti-tumor effects.

Synergistic activities of Panobinostat and doxorubicin in soft tissue sarcomas

BackgroundSoft tissue sarcomas (STS) are rare diseases typically arising from connective tissues in children and adults. However, chemotherapies involved in the treatment of STS may cause toxic side effects and multi-drug chemoresistance, making the treatment even more challenging. Histone deacetylase inhibitors (HDACi) are epigenetic agents which have shown anti-tumor effects as single agent as well as combination use with other drugs. Our project intends to prove the same effects in STS. MethodsPanobinostat (LBH589) plus doxorubicin was selected for investigations based on our previous research. Tumor xenografts were tried in an epithelioid sarcoma model to validate good synergy effects in vivo and a leiomyosarcoma model was used as a negative comparison group. Gene profile changes were studied afterwards. The possible pathway changes caused by HDACi were explored and validated by several assays. ResultsSynergy effect of LBH589 plus doxorubicin was successfully validated in STS cell lines and an epithelioid sarcoma mice model. We tried to reduce the dose of doxorubicin to a lower level and found the drug combination can still inhibit tumor size in mice. Furthermore, gene profile changes caused by LBH589 was studied by RNA-Sequencing analysis. Results showed LBH589 can exert effects on a group of target genes which can regulate potential biological functions especially in the cell cycle pathway.

Abstract 305: Bioinspired synthetic hydrogel in pancreatic cancer organoid matrix modeling

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality with a primary hallmark, including dense collagen matrix in the desmoplastic stroma. Organoid technologies often rely on commercially available EHS mouse sarcoma matrix materials, and it remains uncertain how batch-to-batch variability of this matrix impacts the consistency of cancer signaling. Here, we present a novel photo-activated hydrogel tuned to the properties of the desmoplastic stroma in PDAC organoid cultures. Method: We designed multiple synthetic hydrogel materials with tunable mechanical properties, including SP-139 (Stem Pharm, Inc), a polyethylene glycol-based hydrogel functionalized with norbornene and formed with both degradable and non-degradable crosslinking peptides and a cell adhesion peptide (CRGDS). We used rheometry to measure the elastic modulus of the polymerized hydrogel after visible light photoactivation (395 nm) and expanded multiple cancer organoid cultures. RNAseq was performed from patient-derived xenografts to expanded organoids across matrix designs (Cultrex® v. SP-139) and analyzed by GSVA pathway analyses. Result: We optimized the ratio of non-degradable v. degradable crosslinking peptide to support culture expansion and enzymatic digestion with dispase II (125 µg/mL) and collagenase II (1 mg/mL). Using rheometry, SP-139 at 75% v/v had increased elastic modulus versus Cultrex RGF BME matrix Type 2 at 100% v/v (4992±76.3 v. 68.4±5.6 Pa, p&amp;lt;0.0001). Phenotypic growth was confirmed across serial passages (&amp;gt;8) and a diversity of gastrointestinal cancer types (n=5). Baseline growth rates were identical over 48h between SP-139 (75% v/v) and Cultrex (50% v/v) (+13.4% v. +13.0, p=0.75). Growth remained consistent for SP-139 yet increased with Cultrex at interval of 48-96h (+12.9% SP-139 v +22.4% Cultrex, p&amp;lt;0.005) and interval of 96-144h (+13.6% SP-139 v +24.0% Cultrex®, p&amp;lt;0.0001). Therapeutic response was assessed using 3D CellTiter-Glo® using gemcitabine 100 µM (24h) with comparable inhibition of normalized cell viability in SP-139 v. Cultrex® (35.4±1.9% v. 52.3±6.5%, n.s.) and qualitatively with viability staining using calcein AM and Caspase 3/7FITC. RNAseq revealed Cultrex, when compared to SP-139, yielded down regulation of epoxygenase p450 pathways (padj&amp;lt;0.012) and negative regulation of the p38 MAP kinase cascade (padj&amp;lt;0.048). Conclusions: Functionalized PEG-based synthetic hydrogel matrix materials can be tuned for physical properties to mimic cancer tissue microenvironments more accurately. SP-139, designed to mimic the desmoplastic stroma of the pancreatic cancer microenvironment, has favorable properties including controlled growth rates and compatibility with high content imaging by luminescence and fluorescence. RNAseq pathway analysis shows SP-139 maintains pathways of p450 and MAP kinase signaling of importance with therapeutic resistance. Citation Format: Md Shahadat Hossan, Austin Stram, Ethan Samuel Lin, Sean McIlwain, Connie Lebakken, William Richards, Jeremy D. Kratz. Bioinspired synthetic hydrogel in pancreatic cancer organoid matrix modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 305.

Inhibition of PRL2 Upregulates PTEN and Attenuates Tumor Growth in Tp53-deficient Sarcoma and Lymphoma Mouse Models.

Prl2 deletion attenuates Tp53 deficiency-induced tumor growth by increasing PTEN and reducing Akt activity. Targeting Tp53-null lymphoma with PRL inhibitors lead to reduced tumor burden, providing a therapeutic approach via PTEN augmentation.

Matrigel Tunes H9 Stem Cell-Derived Human Cerebral Organoid Development.

Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition.

Head-to-head comparison of three chelates reveals DOTAGA promising for 225 Ac labeling of anti-FZD10 antibody OTSA101.

To select the most suitable chelate for 225 Ac radiolabeling of the anti-FZD10 antibody OTSA101, we directly compared three chelates: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2,2',2″-(10-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (p-SCN-Bn-DOTAGA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DO3A-NHS-ester). We evaluated the binding affinity of the chelate-conjugated OTSA101 antibodies, as well as the labeling efficiency and stability in murine serum of 225 Ac-labeled OTSA101 as in vitro properties. The biodistribution, intratumoral distribution, absorbed doses, and therapeutic effects of the chelate-conjugated OTSA101 antibodies were assessed in the synovial sarcoma mouse model SYO-1. Of the three conjugates, DOTAGA conjugation had the smallest impact on the binding affinity (p < 0.01). The labeling efficiencies of DOTAGA-OTSA101 and DO3A-OTSA101 were 1.8-fold higher than that of DOTA-OTSA101 (p < 0.01). The stabilities were similar between 225 Ac-labeled DOTA-OTSA101, DOTAGA-OTSA101, and DO3A-OTSA101in serum at 37 and 4°C. The dosimetric analysis based on the biodistribution revealed significantly higher tumor-absorbed doses by 225 Ac-labeled DOTA-OTSA101 and DOTAGA-OTSA101 compared with 225 Ac-DO3A-OTSA101 (p < 0.05). 225 Ac-DOTAGA-OTSA101 exhibited the highest tumor-to-bone marrow ratio, with bone marrow being the dose-limiting tissue. The therapeutic and adverse effects were not significantly different between the three conjugates. Our findings indicate that among the three evaluated chelates, DOTAGA appears to be the most promising chelate to produce 225 Ac-labeled OTSA101 with high binding affinity and high radiochemical yields while providing high absorbed doses to tumors and limited absorbed doses to bone marrow.

Synergistic Antitumor Effect of Combined Radiotherapy and Engineered Salmonella typhimurium in an Intracranial Sarcoma Mouse Model.

Intracranial sarcoma is an uncommon aggressive cancer with a poor prognosis and a high recurrence rate. Although postoperative adjuvant radiotherapy (RT) is the most recommended treatment strategy, it does not significantly improve survival rates. In this study, we used an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (SLpFlaB) as an immunotherapy to assist with the antitumor effects of RT on intracranial sarcoma. In vitro, the expression of γH2AX and cleaved caspase-3 was analyzed by Western blot. In vivo detection of SLpFlaB colonization time in tumors was measured using an in vivo imaging system (IVIS). Tumor growth delay and elimination were demonstrated in an intracranial mouse model, and the distribution of macrophages, M1 macrophages, and CD8+ cells after treatment was measured using FACS analysis. Our findings in vitro suggest that combination therapy increases S-180 radiosensitivity, the expression of DNA double-strand breaks, and programmed cell death. In vivo, combination treatment causes intracranial sarcoma to be eliminated without tumor recurrence and redistribution of immune cells in the brain, with data showing the enhanced migration and infiltration of CD8+ T cells and macrophages, and an increased proportion of M1 macrophage polarization. Compared to RT alone, the combination therapy enhanced the radiosensitivity of S-180 cells, promoted the recruitment of immune cells at the tumor site, and prevented tumor recurrence. This combination therapy may provide a new strategy for treating intracranial sarcomas.

Triterpenoids of Ganoderma lucidum inhibited S180 sarcoma and H22 hepatoma in mice by regulating gut microbiota

In order to explore effect of natural plant extracts on anti-tumor and prevent tumor development. The study assessed the antitumor effect of triterpenoids of Ganoderma lucidum (TGL) on S180 and H22 tumor bearing mice. A triterpene compound, 2α, 3α, 23-trihydroxy-urs-12-en-28-oic acid, was successfully isolated and purified from G. lucidum. S180 and H22 cells were subcutaneously inoculated in the left axilla of mice to establish a transplantable tumor model. After, the mice were orally treated with TGL and evaluated by tumor inhibition rate, organ index, and the serum index. The Bax and Bcl-2 proteins and gut microbiota was analyzed using western blot and 16S rDNA sequencing respectively. The results showed the tumor inhibition rates of TGL were higher than 40% in H22 and S180 tumor bearing mice. TGL had a protective effect on the spleen and thymus, and improved lipid peroxidation caused by the increased free radicals. TGL downregulated Bcl-2 and upregulated Bax. In particular, TGL treatment improved the reduction of gut microbiota richness and structure.

Phase 1 dose-expansion study of oral TP-1287, a cyclin-dependent kinase 9 (CDK9) inhibitor, in patients with Ewing sarcoma (EWS).

TPS11589 Background: Cyclin-dependent kinase 9 (CDK9) blockade inhibits tumor growth and progression by impairing the transcription of key oncogenes, such as myeloid cell leukemia-1 (MCL-1) and c-MYC. CDK9 overexpression has been observed in sarcoma patients (pts), and CDK9 has emerged as a potential therapeutic target in pts with sarcoma. TP-1287 is an investigational orally delivered phosphate prodrug of the CDK9 inhibitor alvocidib. In preclinical studies, TP-1287 has been shown to decrease MCL-1 expression and phosphorylation of RNA polymerase II (RPB1), and inhibit tumor growth in an Ewing sarcoma (EWS) mouse model. Phase 1 dose-escalation in solid tumors has completed and TP-1287 is being investigated in a dose expansion cohort in pts with EWS (NCT03604783). The design of the expansion part of the trial in pts with EWS is herein described. Methods: Up to thirty pts with EWS will be enrolled in this dose expansion cohort. Key eligibility criteria are age ≥18 years (≥12 years, if weight ≥40 kg); histologically confirmed locally advanced or metastatic unresectable EWS; received 1 to 5 prior lines of treatment including an anthracycline; one or more measurable tumors per the RECIST v1.1; ECOG performance status of ≤1; acceptable liver and renal function, and acceptable hematologic and coagulation status; no treatment with surgery, chemotherapy, or investigational therapy within 28 days or 5 half-lives. Eligible pts will be treated with oral TP-1287 monotherapy RP2D established from the dose escalation part (11 mg BID, continuous dosing in a 28-day treatment cycle) and will continue treatment until treatment-related adverse event or disease progression. Assessments will be performed on Day 1 and Day 15 of each cycle. Tumor response assessment will be done after Cycle 2 and at the end of every other cycle thereafter. The primary objectives are objective response rate (ORR) and clinical benefit rate (CBR) ; secondary objectives are median progression-free survival (PFS), PFS rate at 16-weeks and 24-weeks and safety; and exploratory objectives include evaluation of systemic exposure and pharmacodynamics. Safety data will be reviewed on an ongoing basis and a Bayesian approach will be used to assess the efficacy data. Statistical analysis for safety and efficacy parameters will be primarily descriptive in nature. EWS dose expansion cohort is currently recruiting in the United States. Clinical trial information: NCT03604783 .

An OMA1 redox site controls mitochondrial homeostasis, sarcoma growth, and immunogenicity.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

Abstract 5166: Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models

Abstract Advances in immunotherapy have revolutionized cancer treatment, with patients with otherwise incurable disease having long-lasting responses. Unfortunately, deep remissions only occur in a select subset of patients. Cancer progression is common as many current immunotherapies only target one or two pathways, which can quickly lead to tumor resistance. Novel approaches that target several pathways concurrently are therefore needed to promote stronger responses in a wider patient population. Our group has recently discovered that the protein Ubiquitin-specific protease 6 (USP6) upregulates multiple, distinct immunostimulatory pathways. Tumor cell-specific expression of USP6 significantly increases CXCL9, CXCL10, and CCL5 chemokine production that enhances cytotoxic immune cell recruitment. Simultaneously, USP6 also upregulates several ligands and receptors that promote immune cell activation and killing of tumor cells such as CD112, MICA/B, ICAM1, and MHC Class I. In addition, USP6 increases the tumor cell’s sensitivity to immune-derived factors such as Type I and Type II interferons and TRAIL by raising the surface expression of their respective receptors. USP6 is hominid-specific and therefore requires xenografting human tumor cells into athymic, T-cell deficient nude mice. Despite the immunocompromised model, USP6 expression was found to increase immune cell recruitment to the tumor while repolarizing the immune microenvironment toward a more activated, tumor-suppressive state. Strikingly, immune cells in the peripheral blood were also affected with an increase in natural killer cell (NK) activation and a skewing of the myeloid population away from an immunosuppressive phenotype. Our group subsequently demonstrated that a USP6+ tumor was able to inhibit the growth of a USP6− distal tumor, with a concordant increase in NK, macrophage, and dendritic cell activation in the distal USP6− tumor. Given our data, we sought to translate USP6 into a novel immunotherapy. Delivery of mRNA encoding the USP6 protein recapitulates the immunostimulatory effects of USP6 in vitro. Intratumoral (I.T) injection of a lipid nanoparticle (LNP) encapsulating USP6 mRNA significantly inhibited Ewing sarcoma and acute myeloid leukemia growth in athymic mice, despite the model lacking a fully functional immune system. Moreover, I.T injection of USP6 mRNA LNPs inhibited the growth of a non-injected, distal tumor. Altogether, these data support that USP6 is a potentially powerful target and continued development of I.T USP6 mRNA LNPs as a novel immunotherapy. Citation Format: Ian Henrich, Margaret Billingsley, Kanika Jain, Laura Quick, Rob Young, Margaret Chou, Michael Mitchell. Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5166.

Abstract 1877: Tripokin: potential best-in-class for tumor targeted interleukin-2 (IL2) potentiated by tumor necrosis factor (TNF)

Abstract Among the different pharmacological approaches for cancer treatment, antibody-cytokine fusion proteins (also called “immunocytokines”) represent an emerging class of biopharmaceutical products, capable of boosting the immune system to attack tumor cells as a result of a selective accumulation of pro-inflammatory molecules at the site of disease. When used as single agents, immunocytokines are often not able to cure mice and patients. However, it has been shown by our group that the combination of IL2- and TNF- based products was able to completely eradicate tumors in mouse models and to induce complete responses in cancer patients. We have previously described a novel class of immunocytokine products, termed “potency-matched dual cytokine antibody fusion proteins”, consisting of a tumor-targeting antibody (the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin), simultaneously fused to both IL2 and TNF. To match biological activity of the two payloads a single-point mutation was inserted in the TNF sequence and the resulting product, named IL2-F8-TNFmut was able to selectively localize to tumors with excellent tumor-to-organ ratios and it was found to completely eradicate soft-tissue sarcomas in immunocompetent mice, which did not respond to standard chemotherapy. Moreover, the immunocytokine was able to induce complete responses in a variety of mouse models of cancer. The therapeutic activity was further improved when IL2-F8-TNFmut was used in combination with immune checkpoint inhibitors. At the mechanistic level we observed that the anticancer activity was completely abrogated when CD4+ and CD8+ T cells were depleted, while the contribution of NK cells was negligible. We now report the generation of a new potency-matched dual cytokine antibody fusion protein, named Tripokin based on the L19 antibody, specific to the alternatively spliced EDB domain of fibronectin. Compared to the previous product, Tripokin showed a favorable pharmacokinetic profile in monkey, an excellent localization to neoplastic lesions in mice and was easier to formulate for further clinical applications. When tested in tumor bearing mice, Tripokin revealed a superior anti-cancer activity compared to other IL2-based products.The results obtained in this work provided a rationale for future clinical translation activities using Tripokin as potential best-in-class tumor targeted IL2 product. Citation Format: Eleonora Prodi, Riccardo Corbellari, Mattia Matasci, Dario Neri, Roberto De Luca. Tripokin: potential best-in-class for tumor targeted interleukin-2 (IL2) potentiated by tumor necrosis factor (TNF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1877.

Garlicnin B1, an Active Cyclic Sulfide from Garlic, Exhibits Potent Anti-Inflammatory and Anti-Tumor Activities.

This study aimed to investigate the pharmacological activities of garlicnin B1, a cyclic sulfide compound found abundantly in garlic and structurally similar to onionin A1, which has been shown to possess strong anti-tumor effects. In vitro studies demonstrated that garlicnin B1 significantly reduced intracellular reactive oxygen species triggered by hydrogen peroxide in colon cancer cells. In a mouse colitis model induced by dextran sulfate sodium, garlicnin B1 at a low dose (5 mg/kg) remarkably ameliorated the symptoms and pathological progression. Additionally, garlicnin B1 exhibited considerable tumoricidal activity with an IC50 value of ~20 μM, as observed in cytotoxicity assays. In vivo experiments using the mouse sarcoma S180 transplanted model and the azoxymethane (AOM) or DSS-induced colon cancer model showed that garlicnin B1 effectively suppressed tumor growth in a dose-dependent manner, with marked inhibition at 80 mg/kg. These results suggest that garlicnin B1 has diverse functions that could be achieved by carefully manipulating the dosing regimen. We anticipate that garlicnin B1 has the potential to be used beneficially in the future for the treatment of cancer and inflammatory diseases, although further studies are warranted to elucidate its mechanisms of action.

A matrigel-free method for culture of pancreatic endocrine-like cells in defined protein-based hydrogels.

The transplantation of pancreatic endocrine islet cells from cadaveric donors is a promising treatment for type 1 diabetes (T1D), which is a chronic autoimmune disease that affects approximately nine million people worldwide. However, the demand for donor islets outstrips supply. This problem could be solved by differentiating stem and progenitor cells to islet cells. However, many current culture methods used to coax stem and progenitor cells to differentiate into pancreatic endocrine islet cells require Matrigel, a matrix composed of many extracellular matrix (ECM) proteins secreted from a mouse sarcoma cell line. The undefined nature of Matrigel makes it difficult to determine which factors drive stem and progenitor cell differentiation and maturation. Additionally, it is difficult to control the mechanical properties of Matrigel without altering its chemical composition. To address these shortcomings of Matrigel, we engineered defined recombinant proteins roughly 41kDa in size, which contain cell-binding ECM peptides derived from fibronectin (ELYAVTGRGDSPASSAPIA) or laminin alpha 3 (PPFLMLLKGSTR). The engineered proteins form hydrogels through association of terminal leucine zipper domains derived from rat cartilage oligomeric matrix protein. The zipper domains flank elastin-like polypeptides whose lower critical solution temperature (LCST) behavior enables protein purification through thermal cycling. Rheological measurements show that a 2% w/v gel of the engineered proteins display material behavior comparable to a Matrigel/methylcellulose-based culture system previously reported by our group to support the growth of pancreatic ductal progenitor cells. We tested whether our protein hydrogels in 3D culture could derive endocrine and endocrine progenitor cells from dissociated pancreatic cells of young (1-week-old) mice. We found that both protein hydrogels favored growth of endocrine and endocrine progenitor cells, in contrast to Matrigel-based culture. Because the protein hydrogels described here can be further tuned with respect to mechanical and chemical properties, they provide new tools for mechanistic study of endocrine cell differentiation and maturation.

Elevated iNOS and 3′-nitrotyrosine in Kaposi's Sarcoma tumors and mouse model

Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.