Liver Microenvironment Research Articles

Abstract A071: SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Currently, the development of efficacious agents against metastatic castration resistance prostate cancer is hampered by the lack of preclinical models that accurately reflect disease heterogeneity and treatment response. To overcome this barrier, we have developed a preclinical metastasis model using LuCap136 (PDM136), an androgen receptor (AR) positive patient-derived xenograft (PDX) that forms metastases in bone and soft tissues 3 weeks after intracardiac injection. With a combined PTEN −/−, TP53 −/mut, RB1 −/− genotype, PDM136 represents a subgroup of difficult-to-treat mPCRC. PDM136 is organotropic for bone. Mice usually reach the end point after 6-7 weeks due to paraplegia and/or cachexia. Metastatic cells maintain the expression of nuclear AR and epithelial marker cytokeratin 8. Surgical castration significantly inhibits metastatic progression and prolonged the survival rate, however, tumor will eventually progress to castration resistance. To our knowledge, PDM136 is the first AR-positive prostate cancer metastasis model that recapitulates the disease progress of mCRPC. To define the molecular mechanisms of castration resistance in PDM136, we performed RNA expression profiling on bone and soft tissue metastasis collected from intact and castrated mice. Differential expression analysis revealed over 500 transcripts were significantly altered after castration, with SOX2 and ASCL1 among the top up-regulated genes. Gene set enrichment analysis showed decreased AR signaling and increased neuroepithelium transformation in the castrated group. To validate the RNA-seq data, we performed IHC on proteins whose genes were identified by RNAseq including SOX2, ASCL1, as well as androgen receptor (AR), glucocorticoid receptor (GR) and neuroendocrine marker SYP. Surprisingly, we found that the expression of the five markers is organ-specific upon castration. Unsupervised clustering of the IHC stained markers from the bone of castrated mice revealed that AR and GR were undetectable in majority of foci whereas substantial numbers of foci in soft tissue metastasis expressed AR and GR at moderate intensity. Furthermore, we found that AR and GR expression were mutually exclusive in many foci from soft tissue metastasis. SOX2 and ASCL1 were mainly expressed in AR negative tumors, while SYP was expressed in a subpopulation of SOX2+ positive foci. Notably, liver metastasis had the most SYP expressing foci among all organs suggesting liver microenvironment promotes neuroendocrine differentiation. In summary, our IHC results revealed inter-tumor and organ-specific cellular heterogeneity induced by castration. PDM136 is a platform to understand the mechanism of responsible for mCRPC, but also a valuable tool for anti-mCRPC drug discovery. The co-existence of multiple phenotypes indicated that combination therapies targeting multiple pathways are essential to harness this disease. Citation Format: Anson Ku, Juan Yin, Adam G. Sowalsky, Kathleen Kelly. SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A071.

SAT-234 - Immunotherapy resistance in NASH-HCC is driven by the dysfunctional liver microenvironment of NASH

SAT-234 - Immunotherapy resistance in NASH-HCC is driven by the dysfunctional liver microenvironment of NASH

The mechanism of exosome delivered circ-PDE7B promoting liver metastasis of breast cancer.

e13016 Background: According to “seed and soil” hypothesis, tumor microenvironments have major effects on the survival and metastasis of cancers, including breast cancer. Importantly, exosomes are believed to partake in cellular communications through delivering oncogenic ncRNAs (including circRNAs) to the recipient cells and tumor microenvironment, which resulting in distant metastasis. Unfortunately, exosomes delivering circRNAs from breast cancer cells still remained many potential functions that are not completely understood in the progression of liver metastasis of breast cancer. This study aims to provide a theoretical basis to use stable exosomal circRNAs as new biomarkers and target spot for the liver metastasis of breast cancer. Methods: A strategy combining RNA-sequencing technique, RT-qPCR and bioinformatic analysis were used to determine the level of differential expressed circRNAs in tissue exosomes (n=5) and serum exosomes samples (n=5) from liver metastatic breast cancer patients compared with healthy person. The expression of circPDE7B was also detected in tumor tissues (n=10) from liver metastatic patients compared with healthy person, breast cancer cell lines (MDA-MB-231), high-liver metastatic cell lines (LM-MDA-MB-231), exosome from MDA-MB-231 (231/exo) and from LM-MDA-MB-231 (LM-231/exo) by RT-qPCR. In animal experiments, exosomes (enriched in circPDE7B) were injected from tail vein for 24h, follow up with injection with LM-MDA-MB-231 cells, then, fluorescence imaging and pathology showed the metastasis in liver. Results: Isolated serum exosomes were characterization by transmission electron microscope (TEM) and Nanosight 300 (NTA 300) system. We profiled the circRNAs in the tissue and serum exosomes samples from liver metastatic patients and healthy person by RNA sequencing and detected 332 significantly differentially-expressed circRNAs. After bioinformatic analysis with GEO database, circPDE7B was chose to further study. RT-qPCR results showed that higher expression of circPDE7B in liver metastatic tissue and LM-MDA-MB-231, also in LM-231/exo. Moreover, overexpressed circPDE7B in exosomes from breast cancer changes premetastatic microenvironment of the liver microenvironment, which can promote the occurrence of breast cancer liver metastasis. Conclusions: The expression of circPDE7B was found significantly upregulated in tissue and serum exosomes from liver metastatic breast cancer patients. Moreover, circPDE7B could promote liver metastasis of breast cancer. Therefore, this study provides a fresh perspective on novel therapeutic targets and potential biomarkers for liver metastasis from exosomal circRNAs.

The diverse landscape of memory T-cell heterogeneity.

The diverse landscape of memory T-cell heterogeneity.

Wingless/It/β-catenin signaling in liver metastasis from colorectal cancer: A focus on biological mechanisms and therapeutic opportunities.

The liver is the most common site of metastases in patients with colorectal cancer. Colorectal liver metastases (CRLMs) are the result of molecular mechanisms that involve different cells of the liver microenvironment. The aberrant activation of Wingless/It (Wnt)/β-catenin signals downstream of Wnt ligands initially drives the oncogenic transformation of the colon epithelium, but also the progression of metastatization through the epithelial-mesenchymal transition/mesenchymal-epithelial transition interactions. In liver microenvironment, metastatic cells can also survive and adapt through dormancy, which makes them less susceptible to pro-apoptotic signals and therapies. Treatment of CRLMs is challenging due to its variability and heterogeneity. Advances in surgery and oncology have been made in the last decade and a pivotal role for Wnt/β-catenin pathway has been re-cognized in chemoresistance. At the state of art, there is a lack of clear understanding of why and how this occurs and thus where exactly the opportunities for developing anti-CRLMs therapies may lie. In this review, current knowledge on the involvement of Wnt signaling in the development of CRLMs was considered. In addition, an overview of useful biomarkers with a revision of surgical and non-surgical therapies currently accepted in the clinical practice for colorectal liver metastasis patients were provided.

Hepatocellular carcinoma (HCC) tumor microenvironment is more suppressive than colorectal cancer liver metastasis (CRLM) tumor microenvironment

Background and purposeWhile HCC is an inflammation-associated cancer, CRLM develops on permissive healthy liver microenvironment. To evaluate the immune aspects of these two different environments, peripheral blood-(PB), peritumoral-(PT) and tumoral tissues-(TT) from HCC and CRLM patients were evaluated.Methods40 HCC and 34 CRLM were enrolled and freshly TT, PT and PB were collected at the surgery. PB-, PT- and TT-derived CD4+CD25+ Tregs, M/PMN-MDSC and PB-derived CD4+CD25− T-effector cells (Teffs) were isolated and characterized. Tregs’ function was also evaluated in the presence of the CXCR4 inhibitor, peptide-R29, AMD3100 or anti-PD1. RNA was extracted from PB/PT/TT tissues and tested for FOXP3, CXCL12, CXCR4, CCL5, IL-15, CXCL5, Arg-1, N-cad, Vim, CXCL8, TGFβ and VEGF-A expression.ResultsIn HCC/CRLM-PB, higher number of functional Tregs, CD4+CD25hiFOXP3+ was detected, although PB-HCC Tregs exert a more suppressive function as compared to CRLM Tregs. In HCC/CRLM-TT, Tregs were highly represented with activated/ENTPD-1+Tregs prevalent in HCC. As compared to CRLM, HCC overexpressed CXCR4 and N-cadherin/vimentin in a contest rich in arginase and CCL5. Monocytic MDSCs were highly represented in HCC/CRLM, while high polymorphonuclear MDSCs were detected only in HCC. Interestingly, the function of CXCR4-PB-Tregs was impaired in HCC/CRLM by the CXCR4 inhibitor R29.ConclusionIn HCC and CRLM, peripheral blood, peritumoral and tumoral tissues Tregs are highly represented and functional. Nevertheless, HCC displays a more immunosuppressive TME due to Tregs, MDSCs, intrinsic tumor features (CXCR4, CCL5, arginase) and the contest in which it develops. As CXCR4 is overexpressed in HCC/CRLM tumor/TME cells, CXCR4 inhibitors may be considered for double hit therapy in liver cancer patients.

LOXL2 serves as a prognostic biomarker for hepatocellular carcinoma by mediating immune infiltration and vasculogenic mimicry.

The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation. The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM. The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated. LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction.

The role of Th2 in immune surveillance of NAFLD-associated liver metastasis

Abstract Non-alcoholic fatty liver disease (NAFLD) is an increasing global epidemic associated with chronic inflammation, metabolic disorders, immunosuppression and importantly, increased incidence of primary and metastatic liver cancers. We have recently demonstrated that the immunosuppressive nature of NAFLD livers represented by increased myeloid-derived suppressor cells and dysregulated natural-killer T cells, resulted in aggressive tumorigenesis and immunotherapy resistance of primary liver cancer. Given the importance of metabolic and immune imbalance in the formation of metastatic-prone liver microenvironment, we continued to dissect the cellular complexity of NAFLD livers in metastatic liver cancers. Through bulk and single-cell RNA-sequencing (scRNA-seq) dataset analysis from mice and patients with NAFLD or liver metastasis (LM), we found that the CD4 +T helper-2 (Th2) population and the regulon activity of its master transcription factor GATA3were also remarkably reduced in NAFLD or LM compared to healthy controls. Interestingly, increased hepatic metastasis was observed in mice with NAFLD induced by high-fed-high-carbohydrate-diet compared to controlled mice by orthotopic LM mouse models, accompanied with reduced hepatic Th2 cells which negatively correlated with increased triglyceride and cholesterol levels. Taken together, our preliminary findings suggested a potential immunosurveillance role of hepatic Th2 cells that may be dysregulated by lipid metabolic alterations in NAFLD. Therapeutic approaches to restore hepatic Th2 cells may therefore provide new perspective and therapeutic approaches to reduce liver metastasis in the era of NAFLD epidemic. The project is supported by RGC/GRF14104820. The project is supported by RGC/GRF14104820.

Synthetically mannosylated antigens impair the antigen-specific humoral immune response and prolong the therapeutic efficacy of immunogenic protein drugs

Abstract Immunogenic protein drugs trigger an anti-drug antibody (ADA) response in patients which reduces drug efficacy and increases adverse reactions. Our lab has previously shown that targeting antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a novel strategy to further increase delivery of an antigen to the tolerogenic microenvironment of the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production which is maintained throughout subsequent administrations of the native biologic. We found that p(Man) treatment impairs the antibody response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten-skewing or control by Tregs. Through transcriptomic analysis we identified increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment, which may inform the underlying mechanism driving the reduction in ADAs. This modular platform may enhance tolerance to current and future biologics, enabling long-term therapeutic solutions for an ever-increasing healthcare problem.

The tumor microenvironment in the postsurgical liver: Mechanisms and potential targets of postoperative recurrence in human hepatocellular carcinoma.

Surgery remains to be the mainstay of treatment for hepatocellular carcinoma (HCC). Nonetheless, its therapeutic efficacy is significantly impaired by postoperative recurrence, which occurs in more than half of cases as a result of intrahepatic metastasis or de novo tumorigenesis. For decades, most therapeutic strategies on inhibiting postoperative HCC recurrence have been focused on the residual tumor cells but satisfying therapeutic outcomes are barely observed in the clinic. In recent years, a better understanding of tumor biology allows us to shift our focus from tumor cells towardthe postoperative tumor microenvironment (TME), which is gradually identified to play a pivotal role in tumor recurrence. In this review, we describe various surgical stress and surgical perturbation on postoperative TME. Besides, we discuss how such alternations in TME give rise to postoperative recurrence of HCC. Based on its clinical significance, we additionally highlight the potential of the postoperative TME as a target for postoperative adjuvant therapeutics.

Remodeling Collagen Microenvironment in Liver Using a Biomimetic Nano-Regulator for Reversal of Liver Fibrosis.

Liver fibrosis is a progressive histological manifestation that happens in almost all chronic liver diseases. An unabated liver fibrosis may eventually develop into liver cirrhosis or hepatocellular carcinoma. Yet, the strategy for reversal of liver fibrosis is still limited. Herein, a biomimetic nano-regulator (P-ZIF8-cirDNAzyme) is developed to affect both collagen synthesis and degradation in liver to remodel collagen microenvironment. It is found that Zn (II) interference can efficiently inhibit collagen synthesis in activated hepatic stellate cells (aHSC) by inactivating proline 4 hydroxylase and affecting many fibrosis-related signaling pathways. Meanwhile, Zn (II)-dependent circular DNAzymes (cirDNAzymes) are used to efficiently silence tissue inhibitors of metalloproteinase-1, accelerating the degradation of collagen. They act in concert to recover the balance between collagen deposition and degradation. Additionally, ZIF-8-cirDNAzyme is coated by platelet membrane (PM) for precisely targeting aHSC via PM's inflammatory tropism and CD62p-CD44 interaction. In carbon tetrachloride-induced fibrotic mice, P-ZIF-8-cirDNAzyme shows a potent anti-fibrotic effect, greatly reducing the expression of collagen by 73.12% and restoring liver function nearly to normal. This work proposes a prospective platform enabling ion interference and gene silencing, collectively acting in aHSC for reversal of liver fibrosis.

Colorectal cancer liver metastasis: genomic evolution and crosstalk with the liver microenvironment.

Colorectal cancer (CRC) patients frequently develop liver metastases, which are the major cause of cancer-related mortality. The molecular basis and management of colorectal liver metastases (CRLMs) remain a challenging clinical issue. Recent genomic evidence has demonstrated the liver tropism of CRC and the presence of a stricter evolutionary bottleneck in the liver as a target organ compared to lymph nodes. This bottleneck challenging CRC cells in the liver is organ-specific and requires adaptation not only at the genetic level, but also at the phenotypic level to crosstalk with the hepatic microenvironment. Here, we highlight the emerging evidence on the clonal evolution of CRLM and review recent insights into the molecular mechanisms orchestrating the bidirectional interactions between metastatic CRC cells and the unique liver microenvironment.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia

The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

Abstract 6152: Exploiting ferroptosis in mutant BAP1 uveal melanoma

Abstract Uveal melanoma (UM) arises from melanocytes of the uveal tract in the eye and is the most common intraocular malignancy in adults. Primary UM lesions often harbor mutations in G-proteins, GNAQ or GNA11, and are successfully treated by radiotherapy or enucleation. However, 50% of patients develop metastases. Of these metastatic UM (MUM) patients, 80%develop metastases to the liver, an effect strongly correlated with the loss of function of BAP1,a nuclear deubiquitinase. BAP1-mutant MUMs are less responsive to conventional treatments, such as chemotherapy and immune checkpoint inhibitors, with 50% of these patients having an overall survival of only 1 year. We propose that the unique metabolic intermediates found in liver microenvironment may dictate MUM tropism, aggressiveness, and poor therapeutic response. Here, using a combination of in vitro and ex vivo liver slice cultures, we found that the liver microenvironment induces gene expression alterations consistent with limiting the toxicity of reactive oxygen species (ROS). Importantly, we note that mutant BAP1 UM displayed improved ROS detoxification compared to its wildtype counterparts. Furthermore, publicly available datasets suggest patients with mutant BAP1 UM, the subset of patients with liver metastases, have corresponding ROS detoxification gene expression patterns. These findings suggest that the liver microenvironment and BAP1 loss directs MUM to mitigate ROS-associated toxicity. This compensatory mechanism may be therapeutically exploited and pair well as a combination treatment with the recently approved bispecific agent, Tebentafusp, to selectively target liver MUM. Citation Format: Camille J. Cunanan. Exploiting ferroptosis in mutant BAP1 uveal melanoma. [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 6152.

Abstract 5091: Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer

Abstract Since the appearance of oxaliplatin, FOLFOX therapy is positioned as the standard of care for colorectal cancer (CRC). And after the advent of capecitabine, an oral prodrug of 5-FU, both FOLFOX and CAPOX remain the first-line treatment for local and advanced CRC. Even though oral 5-FU prodrug has approved more than two decades ago, orally available oxaliplatin has not been developed yet. Oral chemotherapy has garnered attention in the era of cancer immunotherapy because oral form of the therapy makes metronomic therapy feasible. In this regard, we invented orally absorbable oxaliplatin exploiting complexation with bile acid moiety. The invention encouraged to expand the dosing of combination treatment of 5-FU and oxaliplatin, the most established chemotherapy combination of CRC, into metronomic scheduling. Oral CAPOX therapy is developed to administrate 5-FU and oxaliplatin combination (FOX) metronomically for chemo-immunotherapy. Metronomic dosage successfully induced immunogenic cancer cell death in colorectal cancer xenograft mouse model not exerting toxicity to other organs abolishing immune suppressive effect of chemotherapy. Oral CAPOX boosted tumor immunity translating immune microenvironment. Immune cell population and activation was analyzed using flow cytometry. Suppressive immune cell population decreased after Oral CAPOX treatment. Especially, immune-suppressive macrophage population diminished. Macrophages became immunogenic and presented antigen more frequently. Improved tumor microenvironment combined with anti-PD1 treatment activated and proliferated T cells locally and systemically. Oral CAPOX and anti-PD1 combination therapy activated cytotoxic T cell in tumor tissue and tumor draining lymph node. This therapy also activated NK cells systemically. Oral CAPOX and anti-PD1 combination treatment regressed established tumor completely in more than 90% of the mouse showing its capacity for clinical usage. Liver is the region which makes CRC least immunogenic among metastatic site and the organ CRC is metastasized most abundantly. Immuno-suppresive macrophage in liver is known for the factor discriminating liver metastasized CRC from other organs metastasized one suppressing T cells. Because Oral CAPOX therapy converts macrophage more immunogenic, Oral CAPOX therapy could reverse immunosuppressive tumor microenvironment of liver metastasized colorectal cancer. Oral CAPOX and anti-PD1 combination is treated liver metastasized CRC model and showed standing out antitumor efficacy, and T cell activation. Citation Format: Seong Jin Park, Seho Kweon, Moyo Knowledge Mudhibadhi, Ha Rin Kim, Youngro Byun. Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer. [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 5091.

Abstract 628: Liver macrophages promote breast cancer liver metastasis through migrating neutrophils and initiating NETosis

Abstract Neutrophils extracellular traps (NETs), which are DNA scaffolds in complex with granule proteins, could be detected in the pre-metastatic liver niches and were associated with a poor survival in breast cancer patients. However, the mechanisms of neutrophils infiltration and NETs formation in the pre-metastatic liver tissues remain poorly understood. Here, we dynamically characterized the liver micro-environment of normal and 4T1-bearing BALB/C mouse models with or without liver metastasis by the single cell RNA sequencing. Interestingly, we found that CXCL1 and CXCL2, derived from the liver macrophages, were dramatically elevated in pre-metastatic livers and induced the infiltration of peripheral neutrophils by CXCR2. Besides, liver macrophages were educated by cancer cells-derived cathepsin C to secrete the CXCL1 and CXCL2. Furthermore, we identified that the complement signals were over-activated in pre-metastatic livers and induced neutrophils to NETosis. Therapeutically, targeting CXCL1, CXCL2 and complement signals could inhibit the NET formation and effectively reduce breast cancer liver metastasis. Citation Format: Heliang Li, Linbin Yang, Erwei Song. Liver macrophages promote breast cancer liver metastasis through migrating neutrophils and initiating NETosis [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 628.

Abstract 2270: Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC

Abstract Pancreatic adenocarcinoma (PDAC) is extremely lethal and resistant to checkpoint immunotherapy, characterized by an immunosuppressive tumor microenvironment consisting of stromal and myeloid cells. Prior studies have demonstrated the utility of targeting the chemokine signaling axis CXCR4-CXCL12 (SDF-1), a highlighted feature in PDACs, to overcome the CXCL12-driven immobilization of T cells and thus facilitate their antitumor role within the tumor. Based on these findings, we have conducted a phase 2 trial evaluating the effects of plerixafor, a CXCR4 antagonist, and cemiplimab, a PD1 inhibitor antibody, in patients with metastatic PDAC who have progressed after one line of systemic chemotherapy (NCT04177810). To determine the immunological responses to therapy, blood samples and tissue biopsies were obtained at baseline and during treatment. Hematological assessment confirmed the activity of CXCR4 antagonist in mobilizing hematopoietic precursors (CD34+), immature myeloid cells, and lymphoid cells, as well as monocytic and granulocytic cell populations. Suspension mass cytometry analysis of peripheral blood mononuclear cells revealed that mobilized monocytic subpopulations had high expressions of chemokine receptors CCR2, CCR5, and CXCR2. Histopathologic evaluation of the serial tissue biopsies from the liver metastatic PDAC revealed increased levels of inflammation upon treatment. To further characterize the cellular constituents of the observed inflammation, multiplexed immunohistochemistry by imaging mass cytometry was performed, demonstrating strong trends toward increased infiltration of not only effector T cells but also macrophages and granulocytic cells into the tumor microenvironment. Taken together, these findings suggest that mobilization of myeloid cells by CXCR4 antagonism results in the recruitment of additional myeloid cells from circulation and that alternative chemokine signaling pathways are sufficient for doing so. This implicates a potential mode of resistance against CXCR4-targeted therapies. Furthermore, these observations reinforce the value of ongoing research efforts in the field to subvert the recruitment or immunosuppressive function of myeloid cells, which would be particularly relevant in the setting of CXCR4 antagonism. Citation Format: Sarah M. Shin, Alexei Hernandez, Erin Coyne, Zhehao Zhang, Sarah Mitchell, Jennifer Durham, Xuan Yuan, Hongqui Yang, Elana J. Fertig, Elizabeth M. Jaffee, Katherine M. Bever, Dung T. Le, Won Jin Ho. Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC [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 2270.

Abstract 1313: Hepatic pre-metastatic niche formation by uveal melanoma extracellular vesicles

Abstract Background: Uveal melanoma (UM) is the most common primary intraocular tumor in adults, and half cases develop incurable liver metastases. As the liver is the first and often the only metastatic site of UM, this suggests the existence of a favorable microenvironment for UM cells. Extracellular vesicles (EVs) are released by cancer cells, which allow oncoproteins or genetic material to be transferred to distant cells in order to modify their microenvironment and promote the spread of cancer. Our hypothesis is that EVs from the ocular tumor precondition the hepatic stroma by activating hepatic stellate cells (HSteCs) to promote the colonization by metastatic cells. Our study aims to determine the role of melanomic EVs in the mechanisms governing the remodeling of the liver microenvironment, including the stromal stiffness. Methods: EVs were isolated from UM cells and choroidal melanocytes by differential centrifugation. The mechanisms of EV internalization in HSteCs were investigated by confocal microscopy using inhibitors of endocytosis pathways. The impact of melanomic EVs on the viability, energy metabolism and gene expression profile of targeted cells was studied using MTS assay, LIVE/DEAD assay, and RNA-Sequencing. In addition, activation of the mechanotransduction signaling pathways were studied by immunofluorescence and Western blotting (FAK phosphorylation and YAP nuclear translocation. HSteCs were seeded on hydrogels of varying stiffness (0.8/2.5/8 kPa; which mimic the stiffness of healthy to highly fibrotic liver), and then exposed to EVs to study their level of activation/contractility by confocal microscopy (⍺SMA+) and traction force microscopy. The preconditioning of the liver by melanomic EVs were studied by in vivo fluorescence imaging in immunodeficient mice. The liver was collected, and the EV-driven activation of HSteCs and their remodeling of the hepatic stroma were analyzed by confocal and polarization microscopy. The number of metastatic lesions and the fibrosis were studied by histological staining using Masson's Trichrome and Hematoxylin & Eosin stains. Results: Melanomic EVs increased the proliferation and energy metabolism of HSteCs, and activated pro-fibrotic signaling pathways. The increased rigidity of hydrogels, which mimic advanced pathological stages, favored the internalization of melanomic EVs by HSteCs. The preconditioning of the liver by melanomic EVs promoted the development of metastases and the production of a stiffer extracellular matrix in the animal model. Conclusions: Melanomic EVs preconditioned the liver by establishing a pre-metastatic niche through the activation of stellate cells. A better understanding of the bidirectional crosstalk between metastatic UM cells and stellate cells at the premetastatic and micro-metastatic stages in the liver will allow to identify new molecular mechanisms or biophysical features that could be therapeutically targeted. Citation Format: Kelly Coutant, Léo Piquet, Enola Bollmann, Andrew Mitchell, François Bordeleau, Solange Landreville. Hepatic pre-metastatic niche formation by uveal melanoma extracellular vesicles [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 1313.

Abstract 3636: Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS

Abstract Background: 25% of patients diagnosed with colorectal cancer (CRC) have liver metastasis at presentation, and ~80% of all metastatic CRC are developed in the liver. We previously reported that liver endothelial cells (ECs), a key component of the liver microenvironment, secrete LRG1 to promote CRC growth via activating human epidermal growth factor receptor (ERbB3, also known as HER3). However, we found that LRG1-induced HER3 activation is distinct from the canonical neuregulin 1 (NRG1)-induced HER3 pathway. The present study further validated LRG1 as a new HER3 ligand for promoting mCRC growth and elucidated the novel downstream signaling pathway induced by LRG1-HER3. Methods: We first measured the binding affinity between HER3 and LRG1 by Biolayer interferometry (BLI). We then used in vitro and in vivo xenograft approaches to determine the effect of LRG1 monoclonal antibody (15C4) on HER3 activation and CRC growth. To further determine the role of LRG1 in promoting CRC growth in the liver, we used murine CRC cells in a syngeneic orthotropic liver injection model to establish CRC allografts in the liver of LRG1−/− mice with systemic LRG1 knockout and wild-type siblings (LRG1+/+). We also performed unbiased phospho-MS analysis and subsequent validations to determine the downstream signaling pathway activated by LRG1-HER3. Results: We identified that LRG1 binds to HER3 with the affinity at ~100nM. The LRG1 antibody 15C4 completely attenuated LRG1-induced HER3 activation and in vitro and xenograft growth in vivo. Moreover, LRG1−/− mice with CRC allografts in the liver had 2 times longer overall survival than tumor-bearing LRG1+/+ mice. Lastly, unbiased -omics analysis identified eIF4-protein synthesis is significantly activated by LRG1. With target-specific inhibitors, we further determined that LRG1-HER3 activates the PI3K-PDK1-RSK1/3-eIF4 axis independent of AKT. Conclusions: We identified LRG1 as a novel HER3 ligand and demonstrated that the liver microenvironment-derived LRG1 plays a key oncogenic role in mCRC, by activating a novel RSK-eIF4 survival pathway. Our findings highlighted the potential of blocking LRG1-HER3 and involved downstream pathways for treating patients with mCRC. Citation Format: Moeez Ghani Rathore, Michelle Wright, Wei Huang, Derek Taylor, Yamu Li, Jordan Winter, Zhenghe Wang, John Greenwood, Stephen Moss, Rui Wang. Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS. [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 3636.

Abstract 4456: Inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX2) inhibition reprogram the tumor microenvironment and suppress tumor growth in hepatocellular carcinoma

Abstract Hepatocellular carcinoma is a common cancer worldwide and a leading cause of cancer-related death. Chronic inflammation, accumulation of genetic changes and alternations of the liver microenvironment are critical during the process of carcinogenesis and development of HCC. Inducible nitric oxide synthase (iNOS or NOS2)-derived NO and cyclooxygenase-2 (COX2) are important parts of the neoplastic inflammatory environment. Continuous exposure to moderate to high concentrations of NO, produced by inducible NO synthase (iNOS) and COX2, promotes neoplastic transformation, chemotherapeutic resistance, enhanced cell proliferation, increased inflammation and immune resistance. Our previous study reported that iNOS promoted stem-like characteristics and activation of liver cancer stem cells through Notch signaling pathway. iNOS activity in both the liver cancer stem cells and the microenvironment contributes to tumor progression in mice, suggesting that targeting iNOS in the entire tumor could have therapeutic benefit in HCC. iNOS-derived NO and COX2-derived prostaglandin E2 (PGE2) were shown to promote feed-forward iNOS/COX2 crosstalk because NO induced COX2 and PGE2 induced NOS2. Our research showed that iNOS inhibition with the iNOS inhibitor (1400W) and COX2 inhibitor (Celebrex) diminished HCC tumor growth. The cytokine analysis revealed considerably lower levels of inflammatory cytokines including, IL-6, tissue inhibitor of matrix metalloproteinase-1 (TIMP1), macrophage inflammatory protein-3alpha (MIP-3alpha), IL-1B, CC chemokine subfamily of eosinophil chemotactic proteins eotaxins (CCL11), M-CSF, thymus and activation-regulated chemokine (TARC, CCL17) and leukemia inhibitory factor (LIF) in HCC with iNOS/COX2 inhibition. iNOS/COX2 levels influence the polarization and spatial location of lymphoid cells including CD8+ T cells. Targeting iNOS/COX2 blockade improved CD8+ T cell penetration into the tumor core. We also found that iNOS/COX2 blockade result in more CD4+ T helper cells and CD8+ tumor infiltrating lymphocyte (TIL), but reduce the number of exhausted CD4+ T cells and CD8+ T cells (PD1-high, Lag3+, CD39+ Tex). The results suggesting that iNOS/COX2 inhibitor therapy may alleviate HCC growth by promoting a anti-tumorigenic TME, modifying lymphoid spatial localization and gene expression phenotypes and decreasing T-cell exhaustion. Citation Format: Ronghua Wang, Christof Kaltenmeier, Ruiqi Yang, Tony Haykal, Hamza Yazdani, Celine Tohme, David Geller, Samer Tohme, Timothy Billiar. Inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX2) inhibition reprogram the tumor microenvironment and suppress tumor growth in hepatocellular 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 4456.