Cell Antitumor Immunity Research Articles

Abstract 698: Developing a self-targeted cancer immunotherapy using engineered whole tumor cell-based vaccine

Abstract The administration of inactivated tumor cell lysate is known to induce a potent antitumor immune response, however, their therapeutic efficacy as shown in Phase I-III clinical trials is limited. This could be attributed to the lack of direct cytotoxic effect on tumor cells and the inability to trigger a strong antitumor immune response. Unlike inactivated tumor cells, living tumor cells possess a unique potential to home to and self-target tumors. Therefore, repurposing the tumor cells’ self-homing property and natural source of neoantigens is advantageous for self-targeted cancer immunotherapy. In this study, we developed a genetically engineered living whole cancer cell-based therapeutic with direct tumor killing and immunostimulatory roles. We switched the tumor cells from death ligands sensitive to resistant using CRISPR/Cas9 and subsequently engineered them to release dual cell killing and immunomodulatory agents. These engineered therapeutic tumor cells (ThTC) eliminated established tumor and activating antitumor immune cell trafficking and antigen-specific T cell activation signaling. This mechanism-based efficacy of ThTC translated into a marked survival benefit and long-term immunity in primary, recurrent, and metastatic cancer models in immunocompromised, immunocompetent and humanized mice. Arming naturally neoantigen-rich tumor cells with multidisciplinary therapeutics represents promising cell-based immunotherapy for solid tumors and establishes a roadmap towards clinical translation. Citation Format: Kok-Siong Chen, Natalia Claire Mendonca, Paulo Borges, Khalid Shah. Developing a self-targeted cancer immunotherapy using engineered whole tumor cell-based vaccine [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 698.

Abstract 5167: Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling

Abstract Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [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 5167.

Abstract 2003: Evaluation of liposome encapsulated sunitinib efficacy and immune modulation on renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is one of the deadliest malignant tumors worldwide. The RCC’s standard treatments are including surgery, radiation, and chemotherapy. SUTENT® (sunitinib) was approved by US Food and Drug Administration (FDA) in 2016 to treat advanced RCC. According to the results from the clinical trial, sunitinib is associated with a higher objective response rate and survival rate than IFN-α, which indicates that it is an effective multiple tyrosine kinase inhibitor (TKi) against advanced RCC. Meanwhile, recent studies demonstrated sunitinib could cause tumor immune surveillance by upregulated autophagy to inhibit tumor PD-L1 expression. However, sunitinib targets not only tumor tissue but also multiple organs in the human body, which causes many serious side effects and even leads to death. In order to reduce the toxicity of sunitinib, our group design a novel nanocarrier by PEGylated liposome to encapsulate sunitinib (lipo-sunitinib). The PEGylated liposome could passively target tumor tissue by extravasation of nanoparticles through increased permeability of the tumor vasculature (enhanced permeability and retention, EPR effect). Moreover, the PEGylation on the liposome surface could prolong liposome half-life in the body through increased particle size. Through the EPR effect and PEGylation of lipo-sunitinib to target tumor cells precisely, we successfully increased RCC orthotopic tumor inhibition in mouse models compared with vehicle and sunitinib groups. Furthermore, the RCC tumor immunofluorescence staining revealed lipo-sunitinib had higher activated effector T cells (CD8+IL-2+IFN-γ+) and type 1 dendritic cells (CD11c+CD24+MHCII+) infiltration and lower vessel formation than sunitinib group. The flow cytometry also demonstrated that lipo-sunitinib had increased anti-tumor immune cells and inhibited immune suppressors in the systemic system. More importantly, lipo-sunitinib showed lower toxicity in mouse livers, hearts, and kidneys compared to the sunitinib group. Our results suggested lipo-sunitinib could inhibit tumor growth and prolong survival rate by modulating a positive anti-tumor microenvironment and anti-angiogenesis effects. In sum, with the polish of the delivery system, our novel nanodrug lipo-sunitinib could be a potential new treatment strategy for RCC patients. Citation Format: Po-Fu Yueh, Yuan Chang, Keng-Li Lan, Fei-Ting Hsu. Evaluation of liposome encapsulated sunitinib efficacy and immune modulation on renal cell carcinoma [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 2003.

Abstract 673: p38γ MAPK remodels tumor microenvironment facilitating breast cancer progression and brain metastasis

Abstract Major neoplastic diseases have high incidences of brain metastasis (BrM) which is a clinically dreadful consequence of advanced cancers with an abysmal prognosis. Unfortunately, our understanding of the biology and molecular mechanisms underlying BrM remains rudimentary. Improving our understanding of the pathogenesis of BrM will facilitate the rational development and prioritization of new therapeutic strategies. Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine protein kinases that are key players in various intracellular signaling pathways controlling many cellular processes, e.g., growth, differentiation, stress responses, and immune defense which have a key role in cancer progression. However, little is known regarding the role of MAPKs in the pathogenesis of BrM. Here, we report that p38γ mitogen-activated protein kinase (MAPK12) is overexpressed in multiple tumor types and associated with aggressive breast cancer progression and a poor outcome in BrM patients. Interestingly, we found that the lack of p38γ did not impact the proliferation of tumor cells ex vivo, but significantly reduced both the primary tumor growth and BrM in EO771 and other mammary tumor-bearing immunocompetent mouse models. Our preclinical data indicated that p38γ promoted tumor progression and BrM through remodeling tumor microenvironments in vivo. We have mapped the immune landscape of BrM lesions of EO771-bearing mice using high-dimensional single-cell profiling. Clearly, cytometry by time-of-flight (CyTOF) analysis of the BrM immune infiltrates revealed reprogramming of the immunosuppressive brain microenvironment toward a proinflammatory and antitumoral state with tumor-intrinsic p38γ depletion, as indicated by highly enriched dendritic cell (DC) and cytolytic T cell (CTL) populations in BrM. To explore the impact of p38γ depletion in breast cancer cells, we performed RNA sequencing on EO771 cells having p38γ knocked down versus the control cells. We found that p38γ knocked down led to the upregulation of pathways related to immune cell movement, thereby leading to an increased accumulation of antitumor immune cells in BrM. Together, our findings uncover an important role for tumor-intrinsic p38γ in shaping the brain immune microenvironment and underscore the potential of specific p38γ blockade in enhancing antitumor immunity for the treatment of breast cancer BrM. Citation Format: Xiangliang Yuan, Hao-Nien Chen, Akosua Badu-Nkansah, Yimin Duan, Dihua Yu. p38γ MAPK remodels tumor microenvironment facilitating breast cancer progression and brain metastasis [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 673.

Abstract 2919: A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy

Abstract Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells such as NK cells, from human induced pluripotent stem cells, that can be utilized for cancer immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-5 x 109Jaya cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel method to produce high purity, clinical scale iNK cells and improved the process for the expansion of NK cells efficiently and consistently. This method does not require cell sorting step and the usage of murine derived stromal cells. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature iNK cells in the absence of cell sorting and independent of stroma cells support. Using this novel method, differentiated NK cells could be expanded more than 8,000-fold to enable us to potentially produce 1 x 1012 iNK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply hundreds of doses in clinic from one cGMP manufacturing campaign. iNK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, DNAM-1 etc. Importantly, these iNK cells demonstrate better anti-tumor activities and higher levels of IFNγ and TNFα, compared to primary peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of iNK cells from genetically engineered, clonal master iPSC cell line with knock out of CISH (CISH KO iNK), a key intracellular checkpoint of NK cell anti-tumor immunity. CISH KO iNK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines including K562, Raji, Daudi, SUP-B15, MOLT-4, SKOV-3, OVCAR-4, HCC-827, ZR-75, and BT-474 etc. in vitro and in vivo in an AML xenograft mouse model, compared with unmodified iNK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, creating next-generation NK cell-based immunotherapies. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Gárate-Carrillo, Duygu Ozmadenci, Jaya Thangaraj, Qin Li, Leah Mitchell, Dan S. Kaufman, Robert Hollingsworth, Huang Zhu. A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy [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 2919.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity

The metabolic state represents a major hurdle for an effective adoptive Tcell therapy (ACT). Indeed, specific lipids can harm CD8+ Tcell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 Tcells is superior invitro and invivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

885 Reprogramming the Glioblastoma Immune Microenvironment With Convection Enhanced Gene Therapy Reveals Intratumor IL6 Drives Glioblastoma Immunosuppression and Growth

INTRODUCTION: The glioblastoma microenvironment is an immunosuppressive barrier to therapeutic innovation. METHODS: SB28 or GL261 glioblastoma allografts were implanted into immunocompetent mice and treated with CED of attenuated adeno-associated virus 9 vectors (AAV9) encoding experimental cytokines (Il1b, Ccl4, or Apoa1) underlying infiltration or activation of anti-tumor immune cells in other intracranial tumors. Serial intracranial bioluminescence was used to assess glioblastoma growth and animals were monitored for survival. The impact of gene therapy perturbations on the glioblastoma immune microenvironment was assessed using histology, immunohistochemistry, single-cell mass cytometry (CyTOF), and multiplexed cytokine assays. RESULTS: Serial body weight and systemic multiplexed cytokine measurements showed no evidence of treatment toxicity. AAV9-APOA1 or AAV9-IL1B gene therapy CED treatments attenuated SB28 growth and prolonged survival, decreasing immunosuppressive macrophage infiltration and increasing CD8 T cell and microglia infiltration of the glioblastoma microenvironment compared to control AAV9 vectors. Gene therapy CED treatments did not attenuate GL261 growth or prolong survival, but CyTOF of human glioblastomas (n = 6) in comparison to preclinical models revealed untreated GL261 glioblastomas were endogenously enriched in CD8 T cells and other lymphoid lineages compared to untreated SB28 or human glioblastomas. Multiplexed cytokine assays demonstrated suppression of intratumor IL6 is a conserved mechanism of action underlying glioblastoma gene therapy responses. Analysis of single-cell RNA sequencing of 32,877 cells from human glioblastomas (n = 11) showed IL6 is predominantly produced by radial glial like cancer stem cells or endothelial cells in the tumor microenvironment. In support of these findings, survival from intracranial SB28 glioblastomas was prolonged in Il6 knockout C57BL/6J mice compared to wildtype C57BL/6J mice. CONCLUSIONS: We report a novel strategy using gene therapy and CED to reprogram the glioblastoma immune microenvironment, revealing IL6 drives glioblastoma immunosuppression and growth.

Gelsolin: A comprehensive pan-cancer analysis of potential prognosis, diagnostic, and immune biomarkers.

Introduction: Gelsolin (GSN), a calcium-regulated actin-binding protein, is out of balance in various cancers. It can mediate cytoskeletal remodeling and regulate epithelial-mesenchymal conversion (EMT), but the studies on GSN function in pan-cancer are limited. Methods: We studied the transcription level, prognostic impact, diagnostic value, genetic, epigenetic modification, methylation level and immune significance of GSN in pan-cancer to fully comprehend the function of GSN in various malignancies based on multiple databases like The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Results: Pan-cancer research showed that GSN was downregulated in most tumors and expressed differently in immunological and molecular subtypes of many cancers. GSN had varying impacts on the prognosis of various tumor types. However, all had moderate to high diagnostic efficiency, and serum GSN had good diagnostic value in breast cancer patients (AUC = 0.947). Moreover, GSN was a distinguishing prognosis factor for some specific cancer types. The GSN protein was hypophosphorylated, and its promoter was hypermethylated in most cancers. GSN was linked to the infiltration level of several immunity cells and was essential in anti-tumor immune cell infiltration. KEGG and GSEA analyses showed that GSN was vital in the functions and proteoglycans processes in cancer, chemokine signaling pathway and other immune-related pathways, DNA methylation and cell cycle. Discussion: In conclusion, GSN possesses the ability to be a predictive, diagnostic, and immune indicator in pan-cancer.

Jiawei Yanghe Decoction suppresses breast cancer by regulating immune responses via JAK2/STAT3 signaling pathway

Ethnopharmacological relevanceJiawei Yanghe Decoction (JWYHD) is a widely used traditional Chinese medicine prescription in the clinical setting for the treatment of autoimmune diseases. Many studies showed that JWYHD has anti-tumor activities in cell and animal models. However, the anti-breast cancer effects of JWYHD and the underlying mechanisms of action remain unknown. Aim of studyThis study aimed to determine the anti-breast cancer effect and reveal the underlying mechanisms of action in vivo, in vitro and in silico. Materials and methodsOrthotopic xenograft breast cancer mouse model and inflammatory zebrafish model were used to observe the anti-tumor effect and immune cell regulation of JWYHD. Moreover, the anti-inflammatory effect of JWYHD were evaluated by the expression of RAW 264.7 cells. JWYHD active ingredients were obtained by UPLC-MS/MS and potential targets were screened by network pharmacology. The therapeutic targets and signaling pathways predicted by computer were assessed by Western blot, real-time PCR (RT-PCR), immunohistochemistry (IHC) staining, and Enzyme-linked immunosorbent assays (ELISA) to explore the therapeutic mechanism of JWYHD against breast cancer. At last, Colivelin and Stattic were used to explore the effect of JWYHD on JAK2/STAT3 pathway. ResultsJWYHD significantly decreased the tumor growth in a dose-dependent manner in the orthotopic xenograft breast cancer mouse model. Flow cytometry and IHC results indicated that JWYHD decreased the expressions of M2 macrophages and Treg while increasing M1 macrophages. Meanwhile, ELISA and Western blot results showed a decrease in IL-1β, IL-6, TNFα, PTGS2 and VEGFα in tumor tissue of JWYHD groups. The results were also verified in LPS-induced RAW264.7 cells and zebrafish inflammatory models. TUNEL assay and IHC results showed that JWYHD significantly induced apoptosis. Seventy-two major compounds in JWYHD were identified by UPLC-MS/MS and Network pharmacology. It was found that the significant binding affinity of JWYHD to TNFα, PTGS2, EGFR, STAT3, VEGFα and their expressions were inhibited by JWYHD. IHC and Western blot analysis showed that JWYHD could decrease the expression of JAK2/STAT3 pathway. Furthermore, Colivelin could reverse the decrease effect of JWYHD in vitro. ConclusionJWYHD exerts a significant anti-tumor effect mainly by inhibiting inflammation, activating immune responses and inducing apoptosis via the JAK2/STAT3 signaling pathway. Our findings provide strong pharmacological evidence for the clinical application of JWYHD in the management of breast cancer.

An exosome-related lncRNA signature correlates with prognosis, immune microenvironment, and therapeutic responses in hepatocellular carcinoma

BackgroundExosomes act as essential modulators of cancer development and progression in hepatocellular carcinoma. However, little is known about the potential prognostic value and underlying molecular features of exosome-related long non-coding RNAs. MethodsGenes associated with exosome biogenesis, exosome secretion, and exosome biomarkers were collected. Exosome-related lncRNA modules were identified using PCA and WGCNA analysis. A prognostic model based on data from the TCGA, GEO, NODE, and ArrayExpress was developed and validated. A comprehensive analysis of the genomic landscape, functional annotation, immune profile, and therapeutic responses underlying the prognostic signature was performed on multi-omics data, and bioinformatics methods were also applied to predict potential drugs for patients with high risk scores. qRT-PCR was used to validate the differentially expressed lncRNAs in normal and cancer cell lines. ResultsTwenty-six hub lncRNAs were identified as highly correlated with exosomes and overall survival and were used for prognosis modeling. Three cohorts consistently showed higher scores in the high-risk group, with an AUC greater than 0.7 over time. These higher scores implied poorer overall survival, higher genomic instability, higher tumor purity, higher tumor stemness, pro-tumor pathway activation, lower anti-tumor immune cell and tertiary lymphoid structure infiltration, and poor responses to immune checkpoint blockade therapy and transarterial chemoembolization therapy. ConclusionThrough developing an exosome-related lncRNA predictor for HCC patients, we revealed the clinical relevance of exosome-related lncRNAs and their potential as prognostic biomarkers and therapeutic response predictors.

Metabolic Barriers to Glioblastoma Immunotherapy.

Glioblastoma (GBM) is the most common primary brain tumor with a poor prognosis with the current standard of care treatment. To address the need for novel therapeutic options in GBM, immunotherapies which target cancer cells through stimulating an anti-tumoral immune response have been investigated in GBM. However, immunotherapies in GBM have not met with anywhere near the level of success they have encountered in other cancers. The immunosuppressive tumor microenvironment in GBM is thought to contribute significantly to resistance to immunotherapy. Metabolic alterations employed by cancer cells to promote their own growth and proliferation have been shown to impact the distribution and function of immune cells in the tumor microenvironment. More recently, the diminished function of anti-tumoral effector immune cells and promotion of immunosuppressive populations resulting from metabolic alterations have been investigated as contributory to therapeutic resistance. The GBM tumor cell metabolism of four nutrients (glucose, glutamine, tryptophan, and lipids) has recently been described as contributory to an immunosuppressive tumor microenvironment and immunotherapy resistance. Understanding metabolic mechanisms of resistance to immunotherapy in GBM can provide insight into future directions targeting the anti-tumor immune response in combination with tumor metabolism.

Combining a machine-learning derived 4-lncRNA signature with AFP and TNM stages in predicting early recurrence of hepatocellular carcinoma

BackgroundNear 70% of hepatocellular carcinoma (HCC) recurrence is early recurrence within 2-year post surgery. Long non-coding RNAs (lncRNAs) are intensively involved in HCC progression and serve as biomarkers for HCC prognosis. The aim of this study is to construct a lncRNA-based signature for predicting HCC early recurrence.MethodsData of RNA expression and associated clinical information were accessed from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database. Recurrence associated differentially expressed lncRNAs (DELncs) were determined by three DEG methods and two survival analyses methods. DELncs involved in the signature were selected by three machine learning methods and multivariate Cox analysis. Additionally, the signature was validated in a cohort of HCC patients from an external source. In order to gain insight into the biological functions of this signature, gene sets enrichment analyses, immune infiltration analyses, as well as immune and drug therapy prediction analyses were conducted.ResultsA 4-lncRNA signature consisting of AC108463.1, AF131217.1, CMB9-22P13.1, TMCC1-AS1 was constructed. Patients in the high-risk group showed significantly higher early recurrence rate compared to those in the low-risk group. Combination of the signature, AFP and TNM further improved the early HCC recurrence predictive performance. Several molecular pathways and gene sets associated with HCC pathogenesis are enriched in the high-risk group. Antitumor immune cells, such as activated B cell, type 1 T helper cell, natural killer cell and effective memory CD8 T cell are enriched in patients with low-risk HCCs. HCC patients in the low- and high-risk group had differential sensitivities to various antitumor drugs. Finally, predictive performance of this signature was validated in an external cohort of patients with HCC.ConclusionCombined with TNM and AFP, the 4-lncRNA signature presents excellent predictability of HCC early recurrence.

Mitochondrial C1QBP is essential for T cell antitumor function by maintaining mitochondrial plasticity and metabolic fitness.

The metabolic stress present in the tumor microenvironment of many cancers can attenuate T cell antitumor activity, which is intrinsically controlled by the mitochondrial plasticity, dynamics, metabolism, and biogenesis within these T cells. Previous studies have reported that the complement C1q binding protein (C1QBP), a mitochondrial protein, is responsible for maintenance of mitochondrial fitness in tumor cells; however, its role in T cell mitochondrial function, particularly in the context of an antitumor response, remains unclear. Here, we show that C1QBP is indispensable for T cell antitumor immunity by maintaining mitochondrial integrity and homeostasis. This effect holds even when only one allele of C1qbp is functional. Further analysis of C1QBP in the context of chimeric antigen receptor (CAR) T cell therapy against the murine B16 melanoma model confirmed the cell-intrinsic role of C1QBP in regulating the antitumor functions of CAR T cells. Mechanistically, we found that C1qbp knocking down impacted mitochondrial biogenesis via the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha signaling pathway, as well as mitochondrial morphology via the phosphorylation of mitochondrial dynamics protein dynamin-related protein 1. In summary, our study provides a novel mitochondrial target to potentiate the plasticity and metabolic fitness of mitochondria within T cells, thus improving the immunotherapeutic potential of these T cells against tumors.

Collecting ducts carcinoma: In depth exploration and biologically driven therapy (CICERONE).

TPS754 Background: Collecting duct carcinoma (CDC) is a rare type of non-clear cell renal cell carcinoma with an aggressive course and poor prognosis. The treatment of metastatic disease remains unsatisfactory as no standard-of-care has been established yet. The gap in the clinical management is certainly linked to the poor molecular characterization. To date, a comprehensive biological characterization of CDC and the identification of predictive biomarker is still lacking. Our previous studies proved that CDC is a molecularly heterogeneous disease composed of at least two subtypes distinguished by cell signalling, metabolic and immune-related alterations and featured by a prognostic signature associating T cell immunity and angiogenesis with good clinical outcome and sensitivity to cabozantinib (Gargiuli et al, Cancers 2021 Jun 10;13(12):2903; Todoerti et al, J Clin Oncol 40, no. 16_suppl (June 01, 2022) e16507-e16507; Verzoni et al, Ann Oncol 33:S1218-S121). The aim of CICERONE trial is to change the paradigm of CDC management providing therapies in a biologically informed manner starting from our previous findings. Methods: First, we will define a molecular taxonomy of CDC through comprehensive transcriptomic profiling of 100 CDC samples retrospectively collected within 12 months from the Italian Group of Uropathology Network and the European Union Hub Center. Pathologic review by genitourinary pathologist was required. RNA-Seq will be performed. Results will be merged to that already obtained by the same methodology from the BONSAI dataset (NCT03354884) and evaluated by unsupervised clustering analysis. We will identify differentially expressed genes and pathways. The immune and angiogenic signatures will be searched validating our preliminary results to identify predictive signatures for the subsequent prospective phase II trial. RNA-seq data will be also explored to detect fusion transcripts, as potential source of novel CDC oncogenic drivers, and study immune tumor content through Cybersort deconvolution. RT-qPCR on RNA derived from tumor and normal tissue will be performed to determine tumor specific origin of the fusion transcripts. Immune infiltrate will be also characterized by IHC, together with PDL-1 scoring. Subsequently we will conduct a prospective phase II trial in order to optimize treatment choice and validate prospectively the biological data. In this phase, previously untreated patients with metastatic CDC will be treated according to baseline tumor signature. Moreover, we will assess whether a new concept of immune liquid biopsy that includes the assessment of immunosuppressive vs antitumor immune cells may represent an accessible and compliant tool to test systemic immunity and its implications in response to treatment. This study was supported by the Italian Ministry of health (RF-2019-12369602). Clinical trial information: NCT05372302 .

The Anti-Tumorigenic Role of Cannabinoid Receptor 2 in Colon Cancer: A Study in Mice and Humans.

The endocannabinoid system, particularly cannabinoid receptor 2 (CB2 in mice and CNR2 in humans), has controversial pathophysiological implications in colon cancer. Here, we investigate the role of CB2 in potentiating the immune response in colon cancer in mice and determine the influence of CNR2 variants in humans. Comparing wild-type (WT) mice to CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in aging mice and subsequently used the AOM/DSS model of colitis-associated colorectal cancer and a model for hereditary colon cancer (ApcMin/+). Additionally, we analyzed genomic data in a large human population to determine the relationship between CNR2 variants and colon cancer incidence. Aging CB2-/- mice exhibited a higher incidence of spontaneous precancerous lesions in the colon compared to WT controls. The AOM/DSS-treated CB2-/- and ApcMin/+CB2-/- mice experienced aggravated tumorigenesis and enhanced splenic populations of immunosuppressive myeloid-derived suppressor cells along with abated anti-tumor CD8+ T cells. Importantly, corroborative genomic data reveal a significant association between non-synonymous variants of CNR2 and the incidence of colon cancer in humans. Taken together, the results suggest that endogenous CB2 activation suppresses colon tumorigenesis by shifting the balance towards anti-tumor immune cells in mice and thus portray the prognostic value of CNR2 variants for colon cancer patients.

Integrated clinical analysis and data mining assessed the impact of NOX4 on the immune microenvironment and prognosis of pancreatic cancer.

The tumor microenvironment (TME) of pancreatic cancer is complex. which forms forms a microenvironment with high immunosuppression, ischemia and hypoxia, which promotes tumor proliferation and migration, inhibit the anti-tumor immune response. NOX4 plays an important role in tumor microenvironment and has a significant relationship with the occurrence, development and drug resistance of tumor. Firstly, NOX4 expression in pancreatic cancer tissues under different pathological conditions was detected by applying immunohistochemical staining of tissue microarray (TMA). Transcriptome RNA sequencing data and clinical data of 182 pancreatic cancer samples were downloaded and collated from the UCSC xena database. 986 NOX4-related lncRNAs were filtered by Spearman correlation analysis. prognosis-related NOX4-related lncRNAs and NRlncSig Score were finally obtained by univariate and multivariate Cox regression with Least Absolute Shrinkage and Selection Operator (Lasso) analysis in pancreatic cancer patients. we plotted Kaplan -Meier and time-dependent ROC curves (ROC) to assess the validity in predicting the prognosis of pancreatic cancer. The ssGSEA analysis was applied to explore the immune microenvironment of pancreatic cancer patients as well as to discuss the immune cells and immune status separately. We found that a mature tumor marker, NOX4, play different roles in different clinical subgroups by immunohistochemical analysis and clinical data. Finally, 2 NOX4-related lncRNAs were determined by least absolute shrinkage and selection operator (LASSO) analysis, univariate Cox analysis and multivariate COX analysis. The ROC curve and DCA curve showed that NRS Score had better predictive ability than independent prognosis-related lncRNA and other clinicopathologic indicators. We obtained the relative abundance of 28 infiltrating immune cells by ssGSEA analysis and found a significant positive correlation between the abundance of anti-tumor immune cells and tumor-promoting immune cells in the risk-classified microenvironment. No matter NRS Score or AC092667.2, RP11-349A8.3 was significantly correlated with immune infiltrating cells. Meanwhile, the IC50 of conventional chemotherapeutic agents in high-score group were significantly lower than those in low-score group. As a mature tumor marker, NOX4-related lncRNAs provide new research strategies for prognostic evaluation, molecular mechanism and clinical treatment of pancreatic cancer.

A Polymeric Extracellular Matrix Nanoremodeler for Activatable Cancer Photo‐Immunotherapy

AbstractCancer immunotherapy has shown tremendous potential to train the intrinsic immune system against malignancy in the clinic. However, the extracellular matrix (ECM) in tumor microenvironment is a formidable barrier that not only restricts the penetration of therapeutic drugs but also prevents the infiltration of antitumor immune cells. We herein report a semiconducting polymer‐based ECM nanoremodeler (SPNcb) to combine photodynamic antitumor activity with cancer‐specific inhibition of collagen‐crosslinking enzymes (lysyl oxidase (LOX) family) for activatable cancer photo‐immunotherapy. SPNcb is self‐assembled from an amphiphilic semiconducting polymer conjugated with a LOX inhibitor (β‐aminopropionitrile, BAPN) via a cancer biomarker (cathepsin B, CatB)‐cleavable segment. BAPN can be exclusively activated to inhibit LOX activity in the presence of the tumor‐overexpressed CatB, thus blocking collagen crosslinking and decreasing ECM stiffness. Such an ECM nanoremodeler synergizes immunogenic phototherapy and checkpoint blockade immunotherapy to improve the tumor infiltration of cytotoxic T cells, inhibiting tumor growth and metastasis.

A Polymeric Extracellular Matrix Nanoremodeler for Activatable Cancer Photo-Immunotherapy.

Cancer immunotherapy has shown tremendous potential to train the intrinsic immune system against malignancy in the clinic. However, the extracellular matrix (ECM) in tumor microenvironment is a formidable barrier that not only restricts the penetration of therapeutic drugs but also prevents the infiltration of antitumor immune cells. We herein report a semiconducting polymer-based ECM nanoremodeler (SPNcb) to combine photodynamic antitumor activity with cancer-specific inhibition of collagen-crosslinking enzymes (lysyl oxidase (LOX) family) for activatable cancer photo-immunotherapy. SPNcb is self-assembled from an amphiphilic semiconducting polymer conjugated with a LOX inhibitor (β-aminopropionitrile, BAPN) via a cancer biomarker (cathepsin B, CatB)-cleavable segment. BAPN can be exclusively activated to inhibit LOX activity in the presence of the tumor-overexpressed CatB, thus blocking collagen crosslinking and decreasing ECM stiffness. Such an ECM nanoremodeler synergizes immunogenic phototherapy and checkpoint blockade immunotherapy to improve the tumor infiltration of cytotoxic T cells, inhibiting tumor growth and metastasis.

CXCL9 correlates with antitumor immunity and is predictive of a favorable prognosis in uterine corpus endometrial carcinoma.

The C-X-C motif chemokine ligand-9 (CXCL9) is related to the progression of multiple neoplasms. Yet, its biological functions in uterine corpus endometrioid carcinoma (UCEC) remain shrouded in confusion. Here, we assessed the prognostic significance and potential mechanism of CXCL9 in UCEC. Firstly, bioinformatics analysis of the public cancer database, including the Cancer Genome Atlas / the Genotype-Tissue Expression project (TCGA+ GTEx, n=552) and Gene Expression Omnibus (GEO): GSE63678 (n=7), were utilized for the CXCL9 expression-related analysis in UCEC. Then, the survival analysis of TCGA-UCEC was performed. Futher, the gene set enrichment analysis (GSEA) was carried out to reveal the potential molecular signaling pathway in UCEC associated with CXCL9 expression. Moreover, the immunohistochemistry (IHC) assay of our validation cohort (n=124) from human specimens were used to demonstrate the latent significance of CXCL9 in UCEC. The bioinformatics analysis suggested that CXCL9 expression was significantly upregulated in UCEC patients; and hyper-expression of CXCL9 was related to prolonged survival. the GSEA enrichment analysis showed various immune response-related pathways, including T/NK cell, lymphocyte activation, cytokine-cytokine receptor interaction network, and chemokine signaling pathway, mediated by CXCL9. In addition, the cytotoxic molecules (IFNG, SLAMF7, JCHAIN, NKG7, GBP5, LYZ, GZMA, GZMB, and TNF3F9) and the immunosuppressive genes (including PD-L1) were positively related to the expression of CXCL9. Further, the IHC assay indicated that the CXCL9 protein expression was mainly located in intertumoral and significantly upregulated in the UCEC patients; UCEC with high intertumoral CXCL9 cell abundance harbored an improved prognosis; a higher ratio of anti-tumor immune cells (CD4+, CD8+, and CD56+ cell) and PD-L1 was found in UCEC with CXCL9 high expression. Overexpressed CXCL9 correlates with antitumor immunity and is predictive of a favorable prognosis in UCEC. It hinted that CXCL9 may serve as an independent prognostic biomarker or therapeutic target in UCEC patients, which augmented anti-tumor immune effects to furnish survival benefits.

Single-cell RNA datasets and bulk RNA datasets analysis demonstrated C1Q+ tumor-associated macrophage as a major and antitumor immune cell population in osteosarcoma.

Osteosarcoma is the most frequent primary bone tumor with a poor prognosis. Immune infiltration proved to have a strong impact on prognosis. We analyzed single-cell datasets and bulk datasets to confirm the main immune cell populations and their properties in osteosarcoma. The examples in bulk datasets GSE21257 and GSE32981 from the Gene Expression Omnibus database were divided into two immune infiltration level groups, and 34 differentially expressed genes were spotted. Then, we located these genes among nine major cell clusters and their subclusters identified from 99,668 individual cells in single-cell dataset GSE152048 including 11 osteosarcoma patients. Especially, the markers of all kinds of myeloid cells identified in single-cell dataset GSE152048 were set to gene ontology enrichment. We clustered the osteosarcoma samples in the TARGET-OS from the Therapeutically Applicable Research to Generate Effective Treatments dataset into two groups by complete component 1q positive macrophage markers and compared their survival. Compared with the low-immune infiltrated group, the high-immune infiltrated group showed a better prognosis. Almost all the 34 differentially expressed genes expressed higher or exclusively among myeloid cells. A group of complete component 1q-positive macrophages was identified from the myeloid cells. In the bulk dataset TARGET-OS, these markers and the infiltration of complete component 1q-positive macrophages related to longer survival. Complete component 1q-positive tumor-associated macrophages were the major immune cell population in osteosarcoma, which contributed to a better prognosis.