B16 Tumor Research Articles

Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen spreading and elimination of antigen-negative tumor variants

BackgroundAdoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Target antigen loss resulting either from non-uniform antigen expression or defects in antigen processing and presentation machinery is one well-characterized resistance mechanism. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated enhanced antitumor activity and low systemic exposure in multiple preclinical adoptive T-cell treatment models with homogeneous tumor antigen expression. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants in syngeneic mouse models.MethodsTarget antigen-positive tumors were generated by transducing B16F10 melanoma cells (B16) or Lewis Lung Carcinoma cells (LLC) with a construct expressing the OVA antigen, SIINFEKL, tagged to ubiquitin (B16-U-OVA, LLC-U-OVA), while B16 or LLC tumors served as antigen-negative variants. C57BL/6J mice were subcutaneously injected with heterogeneous tumors composed of 80% B16-U-OVA and 20% B16. Bilateral tumors were established by injecting the left flank with B16-U-OVA or LLC-U-OVA tumors and the right flank injected with B16 or LLC tumors. The tumor-bearing mice then underwent 5.5 Gy total body irradiation, followed by adoptive transfer of OT-I TCR-T cells engineered with or without caIL-12.ResultsTCR-T cells (OT-I) delivered caIL-12 to the B16-U-OVA tumor sites and induced robust tumor control and survival benefits in mice bearing a heterogeneous tumor with OVA-negative variants. caIL-12 exerted its effect on OVA-negative B16 variants primarily by priming and activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-caIL-12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to colocalize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens.ConclusionExpression of caIL-12 by tumor-targeting T cells demonstrated therapeutic effect against target-antigen-negative tumor variants, primarily through the induction of antigen spreading. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors.

Expression of c-erb-B2 oncoprotein as a neoantigen strategy to repurpose anti-neu antibody therapy in a model of melanoma.

In this study, we tested a novel approach of "repurposing" a biomarker typically associated with breast cancer for use in melanoma. HER2/neu is a well characterized biomarker in breast cancer for which effective anti-HER2/neu therapies are readily available. We constructed a lentivirus encoding c-erb-B2, an animal (rat) homolog to HER2/neu. This was used to transfect B16 melanoma in vitro for use in an orthotopic preclinical mouse model, which resulted in expression of rat c-erb-B2 as a neoantigen target for anti-c-erb-B2 monoclonal antibody (7.16.4). The c-erb-B2-expressing melanoma was designated B16/neu. 7.16.4 produced statistically significant in vivo anti-tumor responses against B16/neu. This effect was mediated by NK-cell antibody-dependent cell-mediated cytotoxicity. To further model human melanoma (which expresses < 5% HER2/neu), our c-erb-B2 encoding lentivirus was used to inoculate naïve (wild-type) B16 tumors in vivo, resulting in successful c-erb-B2 expression. When combined with 7.16.4, anti-tumor responses were again demonstrated where approximately 40% of mice treated with c-erb-B2 lentivirus and 7.16.4 achieved complete clinical response and long-term survival. For the first time, we demonstrated a novel strategy to repurpose c-erb-B2 as a neoantigen target for melanoma. Our findings are particularly significant in the contemporary setting where newer anti-HER2/neu antibody-drug therapies have shown increased efficacy.

Joint estimation of compartment-specific T2 relaxation and tumor microstructure using multi-TE IMPULSED MRI.

This study aims to assess how T2 heterogeneity biases IMPULSED-derived metrics of tissue microstructure in solid tumors and evaluate the potential of estimating multi-compartmental T2 and microstructural parameters simultaneously. This study quantifies the impact of T2 relaxation on IMPULSED-derived microstructural parameters using computer simulations and in vivo multi-TE IMPULSED MRI in five tumor models, including brain, breast, prostate, melanoma, and colon cancer. A comprehensive T2 + IMPULSED method was developed to fit multi-compartmental T2 and microstructural parameters simultaneously. A Bayesian model selection approach was carried out voxel-wisely to determine if the T2 heterogeneity needs to be included in IMPULSED MRI in cancer. Simulations suggest that T2 heterogeneity has a minor effect on the estimation of d in tissues with intermediate or high cell density, but significantly biases the estimation of with low cell density. For the in vivo animal experiments, all IMPULSED metrics except are statistically independent on TE. For B16 tumors, the IMPULSED-derived exhibited a notable increase with longer TEs. For MDA-MB-231 tumors, IMPULSED-derived showed a significant increase with increasing TEs. The T2 + IMPULSED-derived of all five tumor models are consistently smaller than . The findings from this study highlight two key observations: (i) TE has a negligible impact on IMPULSED-derived cell sizes, and (ii) the TE-dependence of IMPULSED-derived intracellular volume fractions used in T2 + IMPULSED modeling to estimate and . These insights contribute to the ongoing development and refinement of non-invasive MRI techniques for measuring cell sizes.

Identification of a novel DEC-205 binding peptide to develop dendritic cell-targeting nanovaccine for cancer immunotherapy

Cancer vaccine is regarded as an effective immunotherapy approach mediated by dendritic cells (DCs) which are crucial for antigen presentation and the initiation of adaptive immune responses. However, lack of DC-targeting properties significantly hampers the efficacy of cancer vaccines. Here, by using the phage display technique, peptides targeting the endocytic receptor DEC-205 primarily found on cDC1s were initially screened. An optimized hydrolysis-resistant peptide, hr-8, was identified and conjugated to PLGA-loaded antigen (Ag) and CpG adjuvant nanoparticles, resulting in a DC-targeting nanovaccine. The nanovaccine hr-8-PLGA@Ag/CpG facilitates dendritic cell maturation and improves antigen cross-presentation. The nanovaccine can enhance the antitumor immune response mediated by CD8+ T cells by encapsulating the nanovaccine with either exogenous OVA protein antigen or endogenous gp100/E7 antigenic peptide. As a result, strong antitumor effects are observed in both anti-PD-1 responsive B16-OVA and anti-PD-1 non-responsive B16 and TC1 immunocompetent tumor models. In summary, this study presents the initial documentation of a nanovaccine that targets dendritic cells via the novel DEC-205 binding peptide. This approach offers a new method for developing cancer vaccines that can potentially improve the effectiveness of cancer immunotherapy.

Nitidine chloride inhibits mTORC1 signaling through ATF4-mediated Sestrin2 induction and targets IGF2R for lysosomal degradation

AimsNitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined. MethodsWe explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model. Key findingsNC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts. SignificanceNC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.

Liposomal Phenylephrine Nanoparticles Enhance the Antitumor Activity of Intratumoral Chemotherapy in a Preclinical Model of Melanoma.

Intratumoral injection of anticancer agents has limited efficacy and is not routinely used for most cancers. In this study, we aimed to improve the efficacy of intratumoral chemotherapy using a novel approach comprising peri-tumoral injection of sustained-release liposomal nanoparticles containing phenylephrine, which is a potent vasoconstrictor. Using a preclinical model of melanoma, we have previously shown that systemically administered (intravenous) phenylephrine could transiently shunt blood flow to the tumor at the time of drug delivery, which in turn improved antitumor responses. This approach was called dynamic control of tumor-associated vessels. Herein, we used liposomal phenylephrine nanoparticles as a "local" dynamic control strategy for the B16 melanoma. Local dynamic control was shown to increase the retention and exposure time of tumors to intratumorally injected chemotherapy (melphalan). C57BL/6 mice bearing B16 tumors were treated with intratumoral melphalan and peri-tumoral injection of sustained-release liposomal phenylephrine nanoparticles (i.e., the local dynamic control protocol). These mice had statistically significantly improved antitumor responses compared to melphalan alone (p = 0.0011), whereby 58.3% obtained long-term complete clinical response. Our novel approach of local dynamic control demonstrated significantly enhanced antitumor efficacy and is the subject of future clinical trials being designed by our group.

Abstract LB340: Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice

Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice [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 LB340.

Abstract 7472: Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity

Abstract Introduction: Melanoma patient survival rates have significantly improved recently owing to the development of both targeted therapies and immunotherapies. However, there are still around 50% of patients relapse. The limited response to immunotherapies and global rise in melanoma incidence, with around 290,000 cases newly diagnosed each year, highlight the need for identifying new biomarkers, modulatory pathways, and risk factors for melanoma development. Earlier studies have characterized that aberrant expression of CD47 (a.k.a. “don’t eat me” signal) is one of the mechanisms that tumors exploit to avoid innate immune surveillance. CD47 blockade has been demonstrated to restore myeloid effector function by reinvigorating phagocytosis of tumor cells, which further enhances anti-tumor T cell responses. However, melanoma tumors are resistant to CD47 blockade. Interestingly, we and others have discovered that human and mouse melanoma cells express high levels of SIRPα, the receptor for CD47, which is typically expressed in myeloid compartments. Experimental Procedure: To investigate the role of tumor SIRPα, we designed gRNAs to CRISPR out the SIRPα N-terminal region, where it interacts with CD47, in B16 melanoma and generated SIRPα+/+ (WT) and SIRPα-/- (KO) B16 tumors. We conducted multiple in vitro and in vivo experiments to investigate whether tumor SIRPα played a role in tumor growth and modulated anti-tumor immunity. The preclinical tumor model we used is B16-F10 melanoma and the murine strains we used are the immune-competent (C57BL/6j; B6) and the immune-deficient (RAGnull) mice. For phenotypic and functional assessments, we performed analysis using flow cytometry, immunohistochemistry (IHC) and single cell RNA-sequencing (scRNA-seq) derived from live-sorted CD45+ tumor infiltrating lymphocytes (TILs) of SIRPα-WT and SIRPα-KO B16 tumors. Summary of Unpublished Data Our preliminary data in a preclinical B16-F10 melanoma model revealed that surface SIRPα expression is necessary for optimal B16-F10 melanoma growth in immune-competent mice, but not in immune-deficient RAGnull mice, suggesting a potential crosstalk between tumor SIRPα and the host adaptive immune system. Intriguingly, we found that selective SIRPα deletion in melanoma cells results in a significant increase of immune infiltrates within melanoma tumors that further suppresses tumor growth. Concluding Remarks: SIRPα is required for optimal melanoma development by suppressing anti-tumor adaptive immunity. Ethics Approval: All mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Citation Format: Chien-Huan Weng, Chana Schochet, Linda Hamadene, Shabnam Eghbali, Sébastien Monette, Levi Mangarin, Cailian Liu, Fadi Samaan, Jedd D. Wolchok, Taha Merghoub. Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity [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 7472.

Abstract 4053: Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment

Abstract T cell immune checkpoint blockade (ICB) has shown remarkable promise in melanoma and other cancers. However, most patients do not show clinical benefit. This is because tumors can activate multiple checkpoints and immunosuppressive pathways to evade anti-tumor immune responses. Inhibition of these immune suppressive mechanisms and immune checkpoints or repolarizing the tumor microenvironment (TME) to become more accessible to the immune system may be necessary for maximal therapeutic efficacy of immunotherapies. There is increasing evidence that tumor hypoxia can attenuate the antitumor immune response by promoting immune suppression and inhibiting direct killing by cytotoxic immune cells. We propose that blocking tumor oxygen consumption using drugs that target the mitochondrial complex I (phenformin and metformin) will enhance the efficacy of immunotherapies such as ICB in preclinical models such as B16 melanoma. In this study, we examined how low oxygen levels (hypoxia) influence T cell effector function such as cytotoxic activity and cytokine production in vitro. We co-cultured B16 melanoma cells in the presence of tumor specific T cells (Pmels) and incubated at 37ºC in either normoxia (~21% O2) or hypoxia (3% O2) and assessed killing and intracellular cytokine production 24 - 48 hours later. We show that the ability of tumor antigen-specific T cells to kill tumor cells or make effector cytokine such as IFNγ and TNFα is significantly reduced in hypoxia settings. We also examined the effects of hypoxia targeting drugs (metformin and phenformin) on T cell priming and activation. When T cells were activated in the presence of each drug in vitro, there was enhanced expression of T cell activation markers (CD25, Granzyme B) as well as an increase in their differentiation into central memory T cells. We next determined whether these drugs could alter the TME in vivo by treating C56BL/6J mice bearing established B16 tumors with phenformin and metformin and then examine the effects on the immune system in the tumor, tumor draining lymph nodes and spleen by flow cytometry. We found that these three drugs have differential effects on both the innate and adaptive the immune system in the tumors and periphery. Finally, we tested whether these drugs could delay B16 melanoma growth in vivo as a monotherapy or in combination with anti-PD1. We found that while both drugs can significantly delay tumor growth as monotherapies, only metformin showed improved anti-tumor efficacy when combined with anti-PD-1 in vivo. These studies show that while both drugs target tumor oxygen consumption (via inhibition of complex I), and can delay B16 tumor growth in vivo, they have differential effects on the TME and will need further considerations when combining with immune based therapies such as ICBs. Citation Format: Sadna Budhu, Anais Assouvie, Mamadou Bah, Svena Verma, Inna Serganova, Mayuresh Mane, Juan Zurita, Jason Koutcher, Vladimir Ponomarev, Jedd D. Wolchok, Taha Merghoub. Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment [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 4053.

Abstract 2659: Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [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 2659.

Abstract 6655: Oncolytic adenovirus dually expressing interferon beta and membrane-stable CD40 ligand increases polyfunctional CD8+T cells associated with antitumor activity

Abstract MEM-288 is a dual-transgene armed oncolytic adenovirus in phase 1 clinical development as a standalone agent and in combination with immune checkpoint inhibitors (ICI). It encodes two potent immune agonists: IFNβ and a recombinant membrane-stable chimeric form of CD40L (MEM40). We previously demonstrated this agonist combination is better than either agonist individually to activate conventional dendritic cells type 1 (cDC1) crucial for CD8+ T cell cross-priming, and to increase tumor-antigen reactive CD8+ T cells. Furthermore, first-in-human Phase 1 clinical trial (NCT05076760) results of MEM-288 for advanced non-small cell lung cancer (NSCLC) and other solid tumors showed MEM-288 tumor shrinkage was associated with systemic antitumor T cell immunity as demonstrated by cytokine, T cell clonotype, and tumor neoantigen analysis. Here, we investigated the precise nature of the localized and systemic T cell response induced by MEM40 + IFNβ in preclinical tumor models. Single-cell RNA sequencing of tumor infiltrating T lymphocytes (TILs) revealed that MEM40 + IFNβ elicited a strong increase in T cells expressing Granzyme B and PRF1, key effector cytokines and cytotoxicity mediators. We tracked the CD8+ T cell response in CD45.1 C57BL/6 mice implanted with syngeneic B16 tumor expressing the model antigen OVA. Interestingly, intratumoral treatment with adenovirus encoding MEM40 + IFNβ did not cause an appreciable increase in absolute numbers of antigen-reactive CD8+ T cells, despite tumor growth inhibition. However, MEM40 + IFNβ compared to control unarmed adenovirus induced a significant (p &amp;lt; 0.005) 5-fold increase in polyfunctional IFNγ+TNFα+PRF1+ and IFNγ+TNFα+Granzyme B+ CD8+ T cells. Polyfunctional CD8+ T cells have the capacity to simultaneously produce effector cytokines and cytotoxicity mediators and are known to be strongly associated with antitumor T cell activity. The robust polyfunctional CD8+ T cell response in these studies is consistent with T cell-driven antitumor activity found in both our preclinical tumor experiments and our clinical studies in advanced NSCLC patients responding to MEM-288. Overall, our findings suggest MEM40 + IFNβ generates a strong polyfunctional CD8+ T cell response in the TME that warrants continued evaluation to understand the pleiotropic mechanisms of this cancer immunotherapy. Citation Format: Mark J. Cantwell, Andreas N. Saltos, Hong Zheng, Dana Foresman, Amer A. Beg. Oncolytic adenovirus dually expressing interferon beta and membrane-stable CD40 ligand increases polyfunctional CD8+T cells associated with antitumor activity [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 6655.

Abstract 3610: Utilizing fructose metabolism to fuel anti-tumor immunity

Abstract Consumption of fructose in the form of high fructose corn syrup is elevated in the western diet, but it is not fully understood how fructose metabolism influences anti-tumor immunity. Fructose is canonically metabolized in the intestine and the liver and can shunt into glycolysis. In certain tumor types, fructose metabolism becomes deregulated, allowing tumor cells to metabolize fructose to power their biosynthetic processes. Similarly, reprogramming immune cells to be able to efficiently metabolize fructose may help us power immunotherapy to work in difficult to treat cancers, especially in individuals who have high fructose in their blood or in the tumor microenvironment (TME). Imparting effector T cells with the ability to use fructose for fuel can overcome one of the major metabolic limitations of the TME: limited nutrient supply. As T cells become activated, they alter their metabolism to support growth and proliferation. Activated CD8+ T cells upregulate their glycolysis which results in increased biosynthetic processes, such as synthesis of macromolecules and effector cytokines. However, tumor cells also consume substantial amounts of glucose, resulting in a glucose-depleted microenvironment, which could be one of the major barriers to immunotherapy working in solid tumors. Therefore, we engineered T cells to overexpress a fructose transporter, GLUT5, which is normally not expressed in the immune compartment at high levels. We hypothesized that increased fructose uptake by CD8+ T cells will ameliorate exhaustion caused by the glucose-low tumor microenvironment. Indeed, we determined that GLUT5 T cells are more efficient in killing in low glucose supplemented with fructose. We also observed that GLUT5 expression restores T cell glycolytic capacity under fructose to that of unexhausted T cells under glucose replete conditions. Similarly, GLUT5-expressing T cells proliferate faster in vitro and exhibit an improved ability to control B16 tumor growth in vivo. Additionally, combining GLUT5 cell infusion with checkpoint inhibition resulted in durable tumor control and prolonged mouse survival. Further, engineered CD19-targeted CAR T cells with expression of GLUT5 were more metabolically active and cytotoxic in low glucose conditions, mimicking the TME. In conclusion, GLUT5 expression in effector CD8+ and CAR T cells may allow immunotherapy to function in solid tumors under low glucose conditions. Citation Format: Tanya Schild, Rachel Chirayil, Lyric Haughton, Patrick Wallisch, Marjan Berishaj, David A. Scheinberg, Kayvan R. Keshari. Utilizing fructose metabolism to fuel anti-tumor immunity [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 3610.

Co-delivery of doxorubicin and STING agonist cGAMP for enhanced antitumor immunity

Many chemotherapeutic agents can induce immunogenic cell death (ICD), which leads to the release of danger-associated molecular patterns (DAMPs) and tumor-associated antigens. This process promotes dendritic cells (DCs) maturation and cytotoxic T lymphocyte (CTL) infiltration. However, cancer cells can employ diverse mechanisms to evade the host immune system. Recent studies have shown that stimulator of interferon genes (STING) agonists, such as cGAMP, can amplify ICD-triggered immune responses and enhance the infiltration of immune cells into the tumor microenvironment (TME). Building upon these findings, we constructed a doxorubicin (DOX) and cGAMP co-delivery system (DOX/cGAMP@NPs) for melanoma and triple-negative breast cancer (TNBC) therapy. The results demonstrated that DOX could effectively destroy tumors and induce the release of DAMPs by ICD. Furthermore, in orthotopic 4T1 tumors mice model and subcutaneous B16 tumor mice model, cGAMP could promote the maturation of DCs and CD8+ T cell activation and infiltration by inducing the secretion of type I interferons and pro-inflammation cytokine, which amplified the antitumor immune response induced by DOX. This strategy also promoted the depletion of immunosuppressive cells, potentially alleviating the immunosuppressive TME. In conclusion, our study highlights the combination of DOX-induced ICD and the immune-enhancing properties of cGAMP holds significant implications for future research and clinical applications.

Hemin blocks TIGIT/PVR interaction and induces ferroptosis to elicit synergistic effects of cancer immunotherapy.

The immune checkpoint TIGIT/PVR blockade exhibits significant antitumor effects through activation of NK and CD8+ T cell-mediated cytotoxicity. Immune checkpoint blockade (ICB) could induce tumor ferroptosis through IFN-γ released by immune cells, indicating the synergetic effects of ICB with ferroptosis in inhibiting tumor growth. However, the development of TIGIT/PVR inhibitors with ferroptosis-inducing effects has not been explored yet. In this study, the small molecule Hemin that could bind with TIGIT to block TIGIT/PVR interaction was screened by virtual molecular docking and cell-based blocking assay. Hemin could effectively restore the IL-2 secretion from Jurkat-hTIGIT cells. Hemin reinvigorated the function of CD8+ T cells to secrete IFN-γ and the elevated IFN-γ could synergize with Hemin to induce ferroptosis in tumor cells. Hemin inhibited tumor growth by boosting CD8+ T cell immune response and inducing ferroptosis in CT26 tumor model. More importantly, Hemin in combination with PD-1/PD-L1 blockade exhibited more effective antitumor efficacy in anti-PD-1 resistant B16 tumor model. In summary, our finding indicated that Hemin blocked TIGIT/PVR interaction and induced tumor cell ferroptosis, which provided a new therapeutic strategy to combine immunotherapy and ferroptosis for cancer treatment.

In Vivo and Post-mortem Comparisons of IVIM/Time-dependent Diffusion MR Imaging Parameters in Melanoma and Breast Cancer Xenograft Models.

We aimed to investigate the changes in intravoxel incoherent motion (IVIM) and diffusion parameters between in vivo and post-mortem conditions and the time dependency of these parameters using two different mouse tumor models with different vessel lumen sizes. Six B16 and six MDA-MB-231 xenograft mice were scanned using 7 Tesla MRI under both in vivo/post-mortem conditions. Diffusion weighted imaging with 17 b-values (0-3000 s/mm2) were obtained at two diffusion times (9 and 27.6 ms). The shifted apparent diffusion coefficient (sADC) using 2 b-values (200 and 1500 s/mm2), non-Gaussian diffusion and IVIM parameters (ADC0, K, fIVIM) were estimated at each of the diffusion times. The results were evaluated by repeated measures two-way analysis of variance and post hoc Bonferroni test. In B16 tumors, fIVIM significantly decreased with post-mortem conditions (from 12.6 ± 6.5% to 5.2 ± 1.9%, P < 0.05 at long diffusion time; from 11.0 ± 2.4% to 4.6 ± 2.7%, P < 0.05 at short diffusion time). In MDA-MB-231 tumors, fIVIM also significantly decreased (from 8.8 ± 3.8% to 2.6 ± 1.1%, P < 0.05 at long; from 7.9 ± 5.4% to 2.9 ± 1.1%, P < 0.05 at short). No diffusion time dependency was observed (P = 0.59 in B16 and P = 0.77 in MDA-MB-231). The sADC and ADC0 values tended to decrease and the K value tended to increase after sacrificing and when increasing the diffusion time. The fIVIM values dropped after sacrificing, confirming that IVIM MRI is a promising quantitative parameter to evaluate blood microcirculation. The presence of residual post-mortem fIVIM values suggested that the influence of water molecule diffusion in the blood lumen may contribute to the IVIM effect. Diffusion MRI parameter's time dependency and those changes after sacrificing could possibly provide additional insights into diffusion hindrance mechanisms.

Supramolecular self-assembled gold nanoparticle clusters for synergistic photothermal-chemo tumor therapy.

The combination of photothermal therapy and chemotherapy has emerged as a promising strategy to improve cancer therapeutic efficacy. However, developing a versatile nanoplatform that simultaneously possesses commendable photothermal effect and high drug encapsulation efficiency remains a challenging problem yet to be addressed. Herein, we report a facile supramolecular self-assembly strategy to construct gold nanoparticle clusters (AuNCs) for synergistic photothermal-chemo therapy. By utilizing the functional polysaccharide as a targeted ligand, hyaluronic acid-enriched AuNCs were endowed with targeting CD44 receptor overexpressed on the B16 cancer cells. Importantly, these hyaluronic acid modified AuNCs can shelter therapeutic cargo of doxorubicin (DOX) to aggregate larger nanoparticles via a host-guest interaction with the anchored β-cyclodextrin, as a "nanocluster-bomb" (DOX@AuNCs). The in vitro results revealed that these DOX@AuNCs showed light-triggered drug release behavior and synergistic photothermal-chemo therapy. The improved efficacy of synergistic therapy was further demonstrated by treating a xenografted B16 tumor model in vivo. We envision that our multipronged design of DOX@AuNCs provides a potent theranostic platform for precise cancer therapy and could be further enriched by introducing different imaging probes and therapeutic drugs as appropriate suitable guest molecules.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

Vaccination with post-translational modified, homocitrullinated peptides induces CD8 T-cell responses that mediate antitumor immunity

BackgroundPost-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered...

Microsomal glutathione transferase 1 controls metastasis and therapeutic response in melanoma

While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.

Physalis pubescens L. branch and leaf extracts inhibit lymphoma proliferation by inducing apoptosis and cell cycle arrest.

Physalis pubescens L. is an annual or perennial plant in the family Solanaceae It is used in traditional medicine for treating sore throats, coughs, urinary discomfort, and astringent pain, and externally for pemphigus and eczema in northern China. The proliferation inhibitory activity and mechanisms of the ethyl acetate extract (PHY-EA) from the leaves of Physalis pubescens were investigated. High performance liquid chromatography was used to identify the chemical composition of PHY-EA; sulforhodamine B was used to detect the proliferation inhibitory effect of PHY-EA on MCF-7, CA-46, Hela, HepG2, B16, and other tumor cells; flow cytometry was used to detect the effect of PHY-EA on the lymphoma cell cycle and apoptosis; Western blot was used to detect the expression of the cycle- and apoptosis-related proteins. The expression of Ki-67 and cleaved caspase 3 was detected by immunohistochemistry. The results showed that PHY-EA contained physalin B, physalin O, and physalin L. PHY-EA blocked the cell cycle of G2/M→G0/G1 in lymphoma cells and induced apoptosis in tumor cells. Mouse transplantation tumor experiments showed that PHY-EA had a significant inhibitory effect on mouse transplantation tumors, and the tumor volume and weight were significantly reduced. In conclusion, PHY-EA has a good antiproliferative effect on Burkkit lymphoma, indicating its potential medicinal value.