Mouse Tumor Models Research Articles

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.

Abstract LB076: Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer

Abstract Purpose: Despite receiving great attention as a promising treatment, therapeutic efficacies induced by immune checkpoint inhibitors are low in clinical settings, and thus there are high expectations for the development of innovative diagnostic and therapeutic methods. We previously demonstrated that blocking FSTL1 overcomes anti-PD1 resistance in aged mice. In this study, we comprehensively analyzed and compared gene expressions in splenic T cells harvested from young and old mice receiving anti-FSTL1 or anti-PD1 therapy after tumor implantation using the GeneChip microarray in order to identify a key gene in the anti-PD1 resistance. In addition, we evaluated anti-tumor efficacy induced by blocking the identified molecule using aged tumor models and tumor metastasis models (3LL or Colon26), which are resistant to anti-PD1 therapy. Furthermore, to validate the clinical relevancy, we analyzed immune cells isolated from malignant tumor ascites and peripheral blood cells of gastric cancer patients by flow cytometry. Results: Using the GeneChip microarray data of splenic T cells, we identified α-synuclein (SNCA), which is a neuropathological molecule involved in Parkinson's disease and other synucleinopathies, as a gene that was significantly reduced by anti-FSTL1 therapy, but not anti-PD1 therapy, in splenic T cells of aged tumor models. SNCA+ cell subsets significantly increased not only in CD3+ T cells, but also CD11b+ myeloid cells and CD45- mesenchymal stromal/stem cells, within spleen, peripheral blood, and tumor tissues of aged mice and mouse tumor models, but rarely in those of young and naive mice. Increase of the subsets were also observed in malignant tumor ascites and peripheral blood of gastric cancer patients, but not those of healthy donors. Blocking SNCA with the specific mAb significantly induced potent CD8+ T cells with greater cytotoxicity in the in vitro CTL induction setting, and also significantly induced robust anti-tumor immunity in the in vivo therapeutic setting with anti-PD1-resistant tumor models. Combination of anti-SNCA therapy successfully elicited anti-PD1 therapeutic efficacy, providing significantly better prognosis in many tumor models, including severe tumor ascites models. Conclusions: These results suggest that SNCA is a type of immune inhibitory checkpoint molecules and an immunological determinant of anti-PD1 resistance, although the further study is needed to clarify the molecular functions in immune cells. Targeting this molecule may represent a promising strategy to overcome the anti-PD1 resistance and contribute to improved clinical outcomes in cancer treatment. Citation Format: Chie Kudo-Saito, Hiroshi Imazeki, Hirokazu Shoji, Narikazu Boku. Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB076.

Abstract LB130: Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses

Abstract Immune checkpoint inhibitors have demonstrated significant benefits in treating various types of tumors. However, a considerable number of patients do not respond to this therapy. One proposed mechanism for resistance to immune checkpoint inhibitors is the infiltration of large numbers of regulatory T cells (Tregs) into tumors, which hinders the development of effective antitumor immunity and is often linked to poor prognosis. CCR8 is a G-protein-coupled receptor (GPCR) which is predominantly expressed on Tregs infiltrating tumors. The development of CCR8-specific human antibodies to deplete CCR8+ Tregs is a promising therapeutic strategy for cancer treatment. Here, we report the development and characterization of fully human monoclonal antibodies targeting CCR8 that exhibit potent antibody-dependent cellular cytotoxicity (ADCC) activity. These antibodies were generated in CCR8 knock-out RenMab® mice, which were immunized with native CCR8 antigen prepared via a proprietary cell-free membrane protein synthesis technology. Epitope mapping of the antibodies revealed recognition of a unique epitope on the N-terminus extracellular domain of CCR8. Binding assays indicated cross-reactivity of the antibodies across multiple species; importantly, however, these antibodies did not recognize related chemokine receptors, such as CCR2, CCR4, CCR5, or CX3CR1. In vitro, CCR8 antibodies effectively inhibited calcium flux induced by CCL1 binding to the CCR8 receptor. Furthermore, introduction of DE (S239D, I332E) mutations in the Fc region enhanced ADCC activity of these antibodies. To compare the efficacy of these antibodies with anti-CCR8 benchmarks in vivo, we generated benchmark analogs in-house with the same DE mutations and established syngeneic MC38 tumor models in humanized B-hCCR8 mice. In this assay, our CCR8 antibodies demonstrated potent anti-tumor activity. Notably, one mutant clone with optimized developability demonstrated robust inhibition of tumor growth when compared to both benchmark analogs and its original clone. Taken together, our fully human CCR8 antibodies demonstrate enhanced ADCC function, strong blocking activity, and superior in vivo anti-tumor activity, which may support further development into a novel cancer immunotherapy targeting Tregs. Citation Format: Huichao Liang, Yanan Guo, Meimei Yin, Aki Kawagishi, Takaho Terada, Yoshihiko Kihara, Toru Kanke, Chaoshe Guo, W. Frank An, Yi Yang. Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB130.

Abstract LB428: Discovery of KY-001:A Direct Inhibitor of TEAD-YAP Protein-Protein Interaction for the Treatment of Advanced Solid Tumors with Abnormal Hippo Signaling Pathway

Abstract Background:The Hippo signaling is an important pathway that plays a crucial role in the regulation of numerous biological processes. The binding of YAP/TAZ transcriptional coactivators to TEAD proteins is the core of Hippo signaling in regulating the expression of a variety of genes that mediate many cellular functions. Abnormalities of Hippo signaling including mutation deficiency, fusion and YAP/TAZ overexpression are often found in solid tumors associated with excessive proliferation, drug resistance, or tumor immune evasion. Blocking the Hippo transcription activation, particularly the TAP/TAZ and TEAD interaction, therefore is an attractive therapeutic strategy for the treatment of advanced solid tumors associated with Hippo dysregulation. Results:We have developed a small molecule inhibitor, KY-001, that can bind to TEAD protein and directly disrupt its interaction with YAP/TAZ, thereby blocking Hippo signaling pathway. KY-001 can bind to TEAD (1-4) proteins with strong YAP/TAZ competitive activities in biochemical assays and can inhibit TEAD-YAP mediated transcriptional signals in cell. KY-001 selectively and potently inhibits the proliferation of cancer cells harboring Hippo deficient mutations, such as mesothelioma, lung cancer and other cell lines. Importantly, KY-001 directly affects the expression of some target genes downstream of YAP/TAZ-TEAD transcription activation in cell. KY-001 can strongly inhibit tumor growth in a dose dependent manner against Hippo mutation mesothelioma cancers in two different xenograft mouse tumor models. In addition, KY-001 shows remarkable synergistic effects with EGFR or KRAS G12C inhibitors in mouse non-small lung cancer models, suggesting it may be a promise therapy for treating targeted therapy induced resistant tumors with Hippo dysregulation. Pre-clinical studies shows that KY-001 has good overall druggable profiles with good safety windows in several animal species. Collectively, KY-001 shows high potency and selectivity in vitro and strong anti-tumor activities against Hippo mutation deficient cancers in pre-clinical models. Conclusions:KY-001 is a small molecule drug candidate which directly inhibits the interactions between TEAD and YAP1/TAZ proteins, thereby blocking the transcriptional activation of downstream Hippo signaling. In various tumor models, KY-001 monotherapy shows good efficacy for Hippo mutation deficient tumors. KY-001 also synergizes with other targeted therapy such as EGFR or KRAS G12C inhibitors for treating non-small lung cancers. KY-001 is expected to enter clinical studies in H2, 2024 for the treatment of Hippo dysregulated solid tumors. Citation Format: Kehao Zhao, Yong Zhag, Yufang Shi, Jian Chen. Discovery of KY-001:A Direct Inhibitor of TEAD-YAP Protein-Protein Interaction for the Treatment of Advanced Solid Tumors with Abnormal Hippo Signaling Pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB428.

Abstract LB269: XZP-6924: A potent and highly selective USP1 inhibitor with the potential to improve efficacy and overcome resistance to PARP inhibitors

Abstract Genomic instability exists in most cancer cells, making them more dependent on certain DDR repair pathways. Existing research shows that inhibition of the DNA damage response (DDR) pathway stresses cell replication and survival. Drugs targeting the DDR pathway are effective in the treatment of many types of cancers, such as the approved Poly (ADP-ribose) polymerase (PARP) inhibitors that cause synthetic lethality to the cancers harboring BRCA1/2 mutations and other homologous repair deficiencies (HRD). Although PARP inhibitors have good clinical performance, not all patients respond to PARP inhibitor treatments. In addition, the effectiveness of PARP inhibitors in clinics is also limited by the rise of treatment-related drug resistance. Ubiquitin-specific protease 1 (USP1) is involved in DNA damage repair processes, including translesion synthesis and Fanconi anemia pathways. It has been well documented that USP1 deficiency leads to replication fork instability and synthetic lethality to HRD. Combination of USP1 inhibitors with PARP inhibitors synergistically targets BRCA1/2 mutant cancers, thereby enhancing the antitumor effect and reducing drug resistance to PARP inhibitors.We have developed a potent and highly selective USP1 inhibitor, XZP-6924. The selective inhibition of USP1 by XZP-6924 results in the accumulation of mono-ubiquitinated USP1 substrates, for example, proliferating nuclear antigen (PCNA), leading to DNA double-strand breaks (DSBs) and widespread DNA damage accumulation that contributes to cell death. In vitro data reveal that XZP-6924, when combined with Olaparib (PARPi), displays significantly enhanced activities in multiple Olaparib-resistant HRD tumor cells. In CDX and PDX mouse tumor models, XZP-6924 at low doses demonstrates synergy and superior efficacy with sustained tumor regression when combined with a PARP inhibitor (Olaparib). Furthermore, XZP-6924 possesses favorable ADME properties and pharmacokinetic profiles in preclinical animal species. Preliminary toxicology study data indicate that XZP-6924 is well tolerated with a favorable safety margin.In conclusion, XZP-6924 is a potent and highly selective USP1 inhibitor that has the potential for the combination therapy with PARP inhibitors to increase efficacy and overcome some primary and acquired resistance to PARP inhibitors. Current data strongly support further development of XZP-6924. The IND submission is expected in the second half of 2024. Citation Format: Zhihui Kan, Shujin Bai, Kelin Ma, Bo Chen, Benke Jiang, Feng Wang, Xifeng Ma, Dapeng Zhou, Chunmin Shi, Yanfang Nie, Yunling Wang, Ruxiao Liu, Jun Sun, Huimin Zhou, Mei Xu, Jiakui Li, Bin Liu. XZP-6924: A potent and highly selective USP1 inhibitor with the potential to improve efficacy and overcome resistance to PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB269.

Abstract LB364: Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics

Abstract With the new waves of anti-tumor immunotherapy development, novel immunotherapies based on NK cells have emerged and advanced rapidly. Considering the inherent mechanisms of NK cells, various strategies are employed in tumor immunotherapy to maximize the efficacy of NK cells, including the following: 1) immune checkpoint inhibitors to relieve NK cell inhibition signals; 2) activation of NK cell activity; 3) targeted antibody therapy for tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC), and 4) cell therapy, such as CAR-NK. Although numerous NK-targeting immune therapeutics or various NK-modulating modalities have been developed or advanced toward clinical trials, there is a lack of clinically relevant preclinical animal models that can be widely accepted for the evaluation of NK-modulating agents, particularly for models that involve functional human NK cells in the test system. This study generated a humanized mouse tumor model system which involves specific reconstitution of functional and durable human NK cells in a murine system, providing a clinically relevant tumor microenvironment. We first engineered a stable monoclonal feeder cell line in vitro to expand NK cells isolated from human peripheral blood mononuclear cells (hPBMCs) using magnetic bead separation. Using this approach, the human primary NK cells were robustly and consistently amplified (up to hundreds of fold) while maintaining their purity and functionality. When compared with primary NK cells using in vitro immune assays including ADCC and FACs, the expanded NK cells showed comparable results as the parental cells. In vivo experimental results indicated that a durable and functional NK reconstitution can be established in tumor-bearing severely immune deficient mice expressing hIL-15. Furthermore, this NK-humanized tumor model system was proven to respond to standard of care (SOC) NK-targeting drugs. In summary, this novel NK humanized in vivo model may represent an effective preclinical tool for assessing NK-modulating drugs, with the hope of facilitating the development process of novel immune therapeutics employing both adaptive and innate immunity. Citation Format: Feng Lu, Weiqun Ding, Guangliang Yang, Yaoqing Jia, Zhaoxiang Wang, Xusheng Yuan, Ming Yin, Zhijian Yang, WenQing Yang. Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB364.

Sijunzi decoction enhances sensitivity of colon cancer cells to NK cell destruction by modulating P53 expression

Ethnopharmacological relevanceSijunzi Decoction (SJZD), a traditional Chinese herbal remedy, is frequently employed in the treatment of various cancers, including colon cancer. Previous research suggests that SJZD plays a pivotal role in modulating the immune system and enhancing immunity against tumors. However, the precise role of SJZD in combating colon cancer and its potential molecular functions in regulating natural killer cells remain elusive. Aims of the studyTo elucidate the potential mechanism underlying the anticolon cancer effects of SJZD in synergy with natural killer (NK) cells through both in vivo and in vitro experiments. Materials and methodsIn vivo experiments: A subcutaneous tumor mouse model of colon cancer and in vivo NK cell depletion experiments were conducted to observe the anticolon cancer effects of SJZD. Flow cytometry assessed immune cell depletion in mouse spleens, while immunohistochemical (IHC) staining detected the expression of apoptotic genes in tumor tissues. In vitro experiments: The mechanism by which SJZD regulates the sensitization of colon cancer cells to NK cells was investigated using real-time polymerase chain reaction (RT-PCR), western blotting (WB), and co-culture experiments with NK cells. ResultsSijunzi Decoction (SJZD) significantly impeded tumor growth in mice; however, NK cell depletion markedly attenuated the tumor-suppressive effect of SJZD. Immunohistochemical (IHC) results indicated that SJZD increased the expression of P53, death receptor 4 (DR4), and death receptor 5 (DR5) in tumor tissues. In vitro experiments, 24 h SJZD-pretreated colon cancer cells showed a substantial elevation in P53, DR4, and DR5 levels, and the activity of colon cancer cells significantly diminished after co-culture with NK cells. These effects of SJZD were reversed with the addition of the P53 inhibitor pifithrin-α (PFT-α), resulting in reduced inhibition of colon cancer cells by NK cells. ConclusionSJZD enhances the levels of DR4 and DR5 through the modulation of P53 expression, consequently increasing the sensitivity of colon cancer cells to NK cell-mediated killing. These findings provide a theoretical foundation for the clinical application of SJZD in patients with colon cancer.In this study, we first investigated the effect of SJZD on subcutaneous tumor growth in mice with colon cancer using in vivo assays and assessed the impact of NK cells on the anticolon cancer effect of SJZD in vivo through NK cell depletion. In vitro experiments were conducted to explore the potential mechanism of action of SJZD in NK cell-mediated anticolon cancer effects.

Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment

BackgroundEngineered arenavirus vectors have recently been developed to leverage the body’s immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic...

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient...

Combination low-dose cyclophosphamide with check-point blockade and ionizing radiation promote an abscopal effect in mouse models of melanoma.

The complex strategy of hypo-fractionated radiotherapy (HFRT) in combination with an immune checkpoint inhibitor (ICI) can stimulate a potential systemic antitumor response; however, the abscopal effect is always precluded by the tumor microenvironment, which may limit sufficient T-cell infiltration of distant nonirradiated tumors for certain kinds of inhibitory factors, such as regulatory T-cells (Tregs). Additionally, low-dose cyclophosphamide (LD-CYC) can specifically kill regulatory Tregs and strongly synergize antigen-specific immune responses, which could promote an abscopal effect. We explored whether a triple regimen consisting of HFRT, ICI, and LD-CYC could achieve a better systemic antitumor response in bilateral mouse tumor models. Our data demonstrate that LD-CYC combined with HFRT and antiprogrammed cell death ligand 1 (PDL-1) therapy could enhance the abscopal effect than only HFRT/antiPDL-1 or HFRT alone. Surprisingly, repeat CYC doses cannot further restrain tumor proliferation but can prolong murine overall survival, as revealed by the major pathologic responses. These results are associated with increased CD8 + effector T-cell infiltration, although LD-CYC did not upregulate PDL-1 expression in the tumor. Compared with traditional strategies, for the first time, we demonstrated that a triple treatment strategy remarkably increased the number of radiation-induced tumor-infiltrating CD8 + T-cells, effectively decreasing infiltrating Tregs, and promoting an abscopal effect. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.

PH-responsive self-assembling peptides potentiate therapeutic efficacy via prolonged drug retention and immunomodulation

Insufficient drug accumulation at tumor sites is one of the key factors leading to treatment failure in breast cancer (BC), and developing a chemotherapeutic drug delivery system that can improve the immune microenvironment to expand the benefits of immunochemotherapy for BC remains a challenge. To increase the efficacy of BC treatment by extending drug retention at the tumor site, we developed a pH-responsive peptide modified with the PHSCN peptide sequence (Pep1) that self-assembles to form spherical DM/Pep1 nanoparticles after encapsulating doxorubicin (DOX) and metformin (MET). In the acidic tumor microenvironment, spherical nanocarriers transform into aggregates with a high aspect ratio, facilitating DOX and MET release for combined chemotherapy and immunomodulation. In cellular experiments, this construct provided prolonged drug retention in BC cells. In a subcutaneous tumor mouse model, the DM/Pep1 nanoparticles exhibited a superior tumor inhibition effect compared to that of free DOX/MET. The DM/Pep1 nanocomplex upregulated CD4, induced calreticulin (CRT) exposure, downregulated PD-L1, and enhanced the MET-mediated antitumor immune response. The use of this pH-responsive peptide nanocarrier system with morphological transformation offers a promising strategy for BC therapy.

Knockdown of growth differentiation factor-15 restrains prostate cancer through regulating MAPK/ERK signaling pathway.

Prostate cancer, prevalent among males, is influenced by various molecular factors, including Growth Differentiation Factor 15 (GDF15). Despite its recognized role in multiple tumor types, GDF15's specific involvement in prostate cancer remains insufficiently explored. This study investigates the regulatory function of GDF15 in prostate cancer. To explore GDF15's impact, we established GDF15 knockdown and overexpression models in prostate cancer cells. We quantified mRNA and protein levels using RT-PCR and Western blotting. Functional assays, including CCK8, Transwell, wound healing, and flow cytometry, were employed to evaluate cell proliferation, invasion, migration, and apoptosis. Additionally, the effect of GDF15 on tumor growth was assessed using a metastatic tumor model in nude mice. Elevated GDF15 expression was identified in prostate cancer tissues and cells. The knockdown of GDF15 led to the activation of the MAPK/ERK signaling pathway. C16PAF was found to counteract the inhibitory effects of sh-GDF15 on cell proliferation, invasion, migration, and apoptosis in LNCaP cells. It also reversed the sh-GDF15-induced alterations in the epithelial-mesenchymal transition (EMT) process. In vivo, C16PAF notably mitigated the sh-GDF15-induced suppression of tumor growth. The study demonstrated that sh-GDF15 inhibits cell proliferation, invasion, migration, EMT process, and tumor growth, while it promotes apoptosis. However, these effects were significantly reversed by C16PAF. The study underscores the potential of GDF15 as a target for novel therapeutic interventions in prostate cancer treatment and prevention. These findings illuminate GDF15's multifaceted role in prostate cancer pathogenesis and suggest its viability as a therapeutic target.

Dynamic Proteomic Changes in Tumor and Immune Organs Reveal Systemic Immune Response to Tumor Development

In orthotopic mouse tumor models, tumor progression is a complex process, involving interactions among tumor cells, host cell-derived stromal cells, and immune cells. Much attention has been focused on the tumor and its tumor microenvironment, while the host’s macroenvironment including immune organs in response to tumorigenesis is poorly understood. Here, we report a temporal proteomic analysis on a subcutaneous tumor and three immune organs (LN, MLN, and spleen) collected on Days 0, 3, 7, 10, 14, and 21 after inoculation of mouse forestomach cancer cells in a syngeneic mouse model. Bioinformatics analysis identified key biological processes during distinct tumor development phases, including an initial acute immune response, the attack by the host immune system, followed by the adaptive immune activation, and the build-up of extracellular matrix. Proteomic changes in LN and spleen largely recapitulated the dynamics of the immune response in the tumor, consistent with an acute defense response on D3, adaptive immune response on D10, and immune evasion by D21. In contrast, the immune response in MLN showed a gradual and sustained activation, suggesting a delayed response from a distal immune organ. Combined analyses of tumors and host immune organs allowed the identification of potential therapeutic targets. A proof-of-concept experiment demonstrated that significant growth reduction can be achieved by dual inhibition of MEK and DDR2. Together, our temporal proteomic dataset of tumors and immune organs provides a useful resource for understanding the interaction between tumors and the immune system and has the potential for identifying new therapeutic targets for cancer treatment.

Combination of Molecule-Targeted Therapy and Photodynamic Therapy Using Nanoformulated Verteporfin for Effective Uveal Melanoma Treatment.

Uveal melanoma (UM) is the most common primary ocular malignancy in adults and has high mortality. Recurrence, metastasis, and therapeutic resistance are frequently observed in UM, but no beneficial systemic therapy is available, presenting an urgent need for developing effective therapeutic drugs. Verteporfin (VP) is a photosensitizer and a Yes-Associated Protein (YAP) inhibitor that has been used in clinical practice. However, VP's lack of tumor targetability, poor biocompatibility, and relatively low treatment efficacy hamper its application in UM management. Herein, we developed a biocompatible CD44-targeting hyaluronic acid nanoparticle (HANP) carrying VP (HANP/VP) to improve UM treatment efficacy. We found that HANP/VP showed a stronger inhibitory effect on cell proliferation than that of free VP in UM cells. Systemic delivery of HANP/VP led to targeted accumulation in the UM-tumor-bearing mouse model. Notably, HANP/VP mediated photodynamic therapy (PDT) significantly inhibited UM tumor growth after laser irradiation compared with no treatment or free VP treatment. Consistently, in HANP/VP treated tumors after laser irradiation, the tumor proliferation and YAP expression level were decreased, while the apoptotic tumor cell and CD8+ immune cell levels were elevated, contributing to effective tumor growth inhibition. Overall, the results of this preclinical study showed that HANP/VP is an effective nanomedicine for tumor treatment through PDT and inhibition of YAP in the UM tumor mouse model. Combining phototherapy and molecular-targeted therapy offers a promising approach for aggressive UM management.

GSNOR overexpression enhances CAR-T cell stemness and anti-tumor function by enforcing mitochondrial fitness

Chimeric antigen receptor T cell (CAR-T) has been developed as a promising agent for patients with refractory or relapsed lymphoma and leukemia, but not all the recipients could achieve a long-lasting remission. The limited capacity of in vivo expansion and memory differentiation post activation is one of the major reasons for suboptimal CAR-T therapeutic efficiency. Nitric oxide (NO) plays multifaceted roles in mitochondrial dynamics and T-cell activation, but its function on CAR-T cell persistence and anti-tumor efficacy remains unknown. Herein, we found the continuous signaling from CAR not only promotes excessive NO production, but also suppressed S-nitrosoglutathione reductase (GSNOR) expression in T cells, which collectively led to increased protein S-nitrosylation, resulting in impaired mitochondrial fitness and deficiency of T cell stemness. Intriguingly, enforced expression of GSNOR promoted memory differentiation of CAR-T cell after immune activation, rendered CAR-T better resistance to mitochondrial dysfunction, further enhanced CAR-T cell expansion and anti-tumor capacity in vitro and in mouse tumor model. Thus, we revealed a critical role of NO in restricting CAR-T cell persistence and functionality, and defined that GSNOR overexpression may provide a solution to combat NO stress and render patients with more durable protection from CAR-T therapy.

SIRT5 promote malignant advancement of chordoma by regulating the desuccinylation of c-myc

Chordoma is a relatively rare and locally aggressive malignant tumor. Sirtuin (SIRT)5 plays pivotal roles in various tumors, but the role of SIRT5 in chordoma has not been found. This study was performed to investigate the regulatory effects of SIRT5 on cell proliferation, migration, and invasion and the underlying mechanism in chordoma. A xenograft tumor mouse model was established to assess tumor growth. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA levels of SIRT5 and c-myc. The effects of SIRT5 and c-myc on cell proliferation, migration, and invasion of chordoma cells were detected by cell counting kit-8, colony formation, and Transwell assays. The interaction between SIRT5 and c-myc was evaluated by co-immunoprecipitation (IP) assay. The succinylation of c-myc was analyzed by IP and Western blot. The results showed that SIRT5 expression was upregulated in chordoma tissues and cells. SIRT5 interacted with c-myc to inhibit the succinylation of c-myc at K369 site in human embryonic kidney (HEK)-293T cells. Silencing of SIRT5 suppressed the cell proliferation, migration, and invasion of chordoma cells, while the results were reversed after c-myc overexpression. Moreover, silencing SIRT5 suppressed tumor growth in mice. These findings suggested that SIRT5 promoted the malignant advancement of chordoma by regulating the desuccinylation of c-myc.

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

BackgroundPreviously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. MethodsWe engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/β-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. ResultsHyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating β-catenin activation. Overexpression of EFNA4 activated the β-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity—a phenomenon remediated by the β-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/β-catenin/dCK axis. ConclusionThis study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/β-catenin axis and activation of dCK.

Development of a hypoxia-activated red-emission fluorescent probe for in vivo tumor microenvironment imaging and anti-tumor therapy.

Hypoxia, a significant feature of the tumor microenvironment, is closely associated with tumor growth, metastasis, and drug resistance. In the field of tumor microenvironment analysis, accurately imaging and quantifying hypoxia - a critical factor associated with tumor progression, metastasis, and resistance to therapy - remains a significant challenge. Herein, a hypoxia-activated red-emission fluorescent probe, ODP, for in vivo imaging of hypoxia in the tumor microenvironment is presented. Among various imaging methods, optical imaging is particularly convenient due to its rapid response and high sensitivity. The ODP probe specifically targets nitroreductase (AzoR), an enzyme highly expressed in hypoxic cells, playing a vital role by catalyzing the cleavage of azo bonds. The optical properties of ODP exhibited excellent performance in terms of fluorescence enhancement, fluorescence lifetime (0.81 ns), and detection limit (0.86 µM) in response to SDT. Cell imaging experiments showed that ODP could effectively detect and image intracellular hypoxia and the imaging capability of ODP was studied under various conditions including cell migration, antioxidant treatment, and different incubation times. Through comprehensive in vitro and in vivo experiments, including cellular imaging and mouse tumor models, this work demonstrates the efficacy of ODP in accurately detecting and imaging hypoxia. Moreover, ODP's potential in inducing apoptosis in cancer cells offers a promising avenue for integrating diagnostic and therapeutic strategies in cancer treatment. This innovative approach not only contributes to the understanding and assessment of tumor hypoxia but also opens new possibilities for targeted cancer therapy.

ATF4-SLC7A11-GSH axis mediates the acquisition of immunosuppressive properties by activated CD4+ T cells in low arginine condition

The tumor microenvironment (TME) is restricted in metabolic nutrients including the semi-essential amino acid arginine. While complete arginine deprivation causes Tcell dysfunction, it remains unclear how arginine levels fluctuate in the TME to shape Tcell fates. Here, we find that the 20-μM low arginine condition, representing the levels found in the plasma of patients with cancers, confers Treg-like immunosuppressive capacities upon activated Tcells. Invivo mouse tumor models and human single-cell RNA-sequencing datasets reveal positive correlations between low arginine condition and intratumoral Treg accumulation. Mechanistically, low arginine-activated Tcells engage in metabolic and transcriptional reprogramming, using the ATF4-SLC7A11-GSH axis, to preserve their suppressive function. These findings improve our understanding of the role of arginine in human Tcell biology with potential applications for immunotherapy strategies.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models [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 4074.