Mouse Model Of Liver Metastasis Research Articles

Abstract PO5-25-01: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

Abstract PO3-26-08: Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination

Abstract More than half of patients with stage IV breast cancer will eventually develop liver metastasis. Due to their nature, liver metastases are hard to treat, and the five-year survival rate for stage IV breast cancer is very low (~11%). In the recent years, vital roles macrophages play in cancer progression and immune evasion have become increasingly clear, making them an appealing target for therapeutic intervention. As one of the most abundant cell populations recruited to the tumor microenvironment, tumor-associated macrophages (TAM) are typically immunosuppressive Arg1+ M2-like macrophages, which support cancer growth, metastasis progression, and drug resistance. Numerous studies, including from our laboratory, have shown that reversal of the macrophage phenotype to the classical inflammatory CD80+ M1-like macrophages is very promising for cancer eradication. In this study, we used CRISPR loaded into lipid nanoparticles (CRISPR-LNP) to modulate macrophage polarization in breast cancer metastasis in vivo. We hypothesize that CRISPR-mediated gene editing through lipid nanoparticle (LNP) delivery of CRISPR enzyme and target gRNA (CRISPR-LNP) is a highly effective and safe approach. Using our CRISPR-LNP platform, we screened multiple genes with significant roles in macrophage polarization and found that targeting the RICTOR gene alone enabled reprograming of pro-tumorigenic M2 TAMs to anti-tumorigenic M1 phenotype in vitro and in vivo. We administered the CRISPR-LNP targeting RICTOR (CRISPR-RIC-LNP) on a syngeneic liver metastasis mouse model via intravenous injection. Single dose studies were conducted to determine biodistribution, treatment efficacy, and shift in immune landscape of the tumor after treatment via immunofluorescence staining, Imaging Mass Cytometry, and single cell RNA sequencing. A spatial map was built using digital pathology analysis for investigation of the interactions amongst immune and tumor cells within the tumor immune microenvironment. In 4T1 breast cancer liver metastatic model, we observed that CRISPR-RIC-LNP treatment: (i) accumulated in the liver metastases lesions in vivo 24 hours after injection. ~0.5% of total injected dose, which corresponds to ~75% of fluorescence from the whole body at 24h time point was confined only in the metastatic regions; (ii) led to modest increase of macrophage number, and (iii) significant shift of macrophage phenotype from immunosuppressive, pro-tumorigenic Arg1+, CD206+ macrophages to inflammatory, anti-tumor CD80+ phenotype (from ~5 to ~9% of total cell in the tumor lesions) 24 hours after injection; (iv) reduced the cancer cell number by almost 80%; and (v) dramatically reduced exhausted T-cell and regulatory T-cell population, as determined by single cell RNA sequencing analysis. We plan to validate our preliminary finding with flow cytometry analysis of metastatic lesions, and evalute the efficacy of CRISPR-RIC-LNP as single and in combinatorial therapies in survival studies. These data showed that reprograming macrophages from cancer-supporting to cancer-eliminating phenotype is a promising tool to suppress cancer cell growth and increase the presence of cytotoxic CD8+ T-cells. We propose that this approach can be efficiently used as single therapy or as combination with immune checkpoint inhibitors to obtain synergistic effects and efficiently eradicate cancer cells. We will assess the safety, optimize the accumulation in TME, and devise methods to limit exposure to healthy cells. In the future, we also aim to expand scope of our candidate therapy as therapeutics for other types of breast cancer metastases. Citation Format: Fransisca Leonard, Tyler Galbraith, Nourhan Abdelfattah, Arturas Ziemys, Chihiro Hashimoto, Harlan Cook, Yitian Xu, Shu-Hsia Chen, Roberto Rosato, Kyuson Yun. Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-08.

SDCBP modulates tumor microenvironment, tumor progression and anti-PD1 efficacy in colorectal cancer.

Anti-programmed cell death 1 (aPD1) therapy has yielded limited success in patients with colorectal cancer (CRC). Syndecan binding protein (SDCBP), encodes a PDZ domain-containing protein that is essential for cellular processes, including cell adhesion, migration, and signal transduction. Here, we investigated the effect of SDCBP on tumor progression, immunotherapy, and the tumor microenvironment (TME) in CRC. High expression of SDCBP is associated with non-response to immunotherapy and correlated with poorer disease-free survival (DFS) in CRC patients. Inhibiting SDCBP by transfecting shRNA or using its inhibitor zinc pyrithione (ZnPT) hindered proliferation and metastasis while enhancing the efficacy of aPD1 treatment in a mouse xenograft model and liver metastasis model. The TME of CRC was significantly altered following ZnPT treatment characterized by a reduced amount of M2 macrophages and a heightened percentage of M1 macrophages. The co-culture system of CRC cells and macrophages provided evidence that SDCBP silencing promoted the repolarisation of M2 macrophages into M1. SDCBP promotes the proliferation, metastasis, and immunotherapy resistance of CRC. Thus, ZnPT represents an effective SDCBP inhibitor and exhibits considerable potential for combination with aPD1 to enhance immunotherapy efficacy.

Abstract 4125: Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis

Abstract Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. LIN28B, an RNA-binding protein that regulates mRNA translation, has garnered significant interest in recent cancer research. Our lab has established that LIN28B is an oncogene and promotes CRC metastasis. However, the exact mechanisms through which LIN28B exerts these effects remain elusive. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is altered in 50-70% of CRCs and is characterized by PIK3CA gene mutations in 20-30% of CRC cases. PIK3CA encodes the catalytic subunit p110α of the PI3Kα enzyme, which is critical for PI3K/AKT signaling. Activation of this pathway is associated with worse CRC prognosis. Among the components of this signaling cascade, the ribosomal protein S6 kinase (S6K) is a crucial downstream effector, playing a pivotal role in protein synthesis and cell growth. Despite the frequent occurrence of PIK3CA mutations in CRC, targeted therapies for PIK3CA-mutant CRC are lacking. Alpelisib, a PI3Kα-specific inhibitor approved by the FDA for a subset of PIK3CA-mutant breast cancers, remains largely unassessed in CRC. We hypothesize that LIN28B-mediated CRC metastasis is facilitated by the activation of the PI3K/AKT pathway, representing a potential target for Alpelisib or other related inhibitors. Results: Our data indicate that LIN28B expression in CRC cells promotes liver metastasis in mouse models of metastatic CRC (mCRC). Furthermore, LIN28B expression activates the PI3K/AKT pathway, as measured by an AKT phosphorylation array. Treatment of CRC cells with SC79, a pan-AKT activator, enhanced cell migration and invasiveness. Similarly, VillinCre;R26Pik3ca transgenic mice, expressing a constantly active mutant form of PI3Kα in the intestinal epithelium, demonstrated crypt hyperplasia and an amplified propensity for tumorigenesis, mirroring the effects of LIN28B expression in CRC. Importantly, Alpelisib effectively hinders LIN28B-driven CRC progression in cell lines, transgenic mice-derived organoids, and mCRC mouse models, as well as the growth rate of CRC patient-derived organoids (PDOs). The effects observed with Alpelisib treatment were replicated with inhibition of S6K using either LY25284702 or PF4708671. Moreover, a combination of low-dose Alpelisib with low-dose LY25284702 or PF4708671 synergistically restricted the growth of PDOs. Conclusion: Our data provide compelling evidence that the PI3K/AKT pathway regulates LIN28B-mediated CRC metastasis, and its inhibition by Alpelisib is feasible. This underscores the potential for precision medicine in CRC, offering targeted therapies to PIK3CA-mutant CRC patients who lack specific treatment options. Furthermore, our study highlights the clinical advantages of combined inhibition strategies for improved anti-metastatic outcomes, a field in dire need of interventions in mCRC. Citation Format: Alice E. Shin, Kensuke Sugiura, Secunda W. Kariuki, David A. Cohen, Christopher J. Lengner, Peter A. Sims, Joel T. Gabre, Anil K. Rustgi. Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis [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 4125.

Abstract A114: Low-dose carbon monoxide halts pancreatic cancer metastasis

Abstract Pancreatic cancer is projected to become the second leading cause of cancer-related death by 2030. Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of pancreatic cancer cases. Over the last 30 years, standard care of chemotherapy only extends the survival of metastatic pancreatic cancer patients for a few months. Despite years of remarkable innovations in cancer treatment, the 5-year survival rate of PDAC has remained dismal. The recalcitrant nature of PDAC to available treatments highlights an urgent need for novel therapeutics in order to substantially increase survival. Carbon monoxide (CO) is produced endogenously in humans and has been established as an important, biologically active signaling molecule. Accumulating evidence suggests the therapeutic applications of safe, low-dose CO for various diseases. We evaluate the therapeutic effect of CO on pancreatic cancer progression. We show that 250 ppm CO inhibits migration of pancreatic cancer. We demonstrate that low-dose CO suppresses metastasis of PDAC in an orthotopic mouse model of liver metastasis and a spontaneous PDAC metastasis mouse model after the removal of primary tumor xenograft tumors. Mechanistically, low-dose CO blocks transcription of heme importers, leading to diminished intracellular heme levels and a heme-regulated enzyme, cytochrome P4501B1 (CYP1B1). Either supplementing heme or overexpressing CYP1B1 reverses the anti-migration effect of low-dose CO. Altogether, low-dose CO therapy inhibits pancreatic cancer cell migration, prevents disseminated cancer cells from expanding into gross metastases, and improves survival in pre-clinical mouse models of metastasis. Citation Format: Tiantian Zhang, George Zhang, Xiang Chen, Zhengming Chen, Adrian Y. Tan, Anthony Lin, Cheryl Zhang, Guoan Zhang, Jenny Xiang, Erika Hissong, Yao-Tseng Chen, Nancy Du. Low-dose carbon monoxide halts pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A114.

Abstract C103: Differential acquisition of extracellular lipid correlates with pancreatic cancer subtype and metastatic tropism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease, and the third leading cause of cancer-related death with a 5-year overall survival rate of less than 5%. Recent studies have identified two major subtypes of pancreatic cancer, basal and classical, that are predictive of patient prognosis, with basal tumors corresponding with more aggressive disease. While transcriptional signatures define basal and classical tumors and cell lines, little is known regarding the metabolic vulnerabilities linked to the basal versus classical state. Using unbiased computational interrogation to uncover metabolic genes correlating with known markers of classical PDAC, we identified high expression of Proprotein convertase subtilisin/kexin type-9 (PCSK9) – a negative regulator of receptor mediated uptake of cholesterol containing low-density lipoprotein (LDL) – as correlating with several established markers of classical PDAC. In contrast, PCSK9 expression is suppressed in Basal PDAC. Accordingly, basal PDAC uptake high levels of LDL, are sensitive to LDL depletion and show higher dependence on cholesterol uptake, relative to classical PDAC. As lipoproteins are produced in the liver, we hypothesized that basal PDAC cells may more efficiently seed and grow within the liver. Consistent with this hypothesis, low PCSK9 expression is correlated with liver metastasis in mouse models of PDAC and patient PDAC specimens. Ongoing studies will interrogate the role of PCSK9 and cholesterol uptake and utilization in metastatic tropism, and uncover unique features and vulnerabilities of the most aggressive variant of PDAC. Citation Format: Gilles Rademaker, Grace A. Hernandez, Longhui Qiu, Kwun W. Wen, Aakriti Jain, Grace A. Kim, Roberto Zoncu, Rushik M. Perera. Differential acquisition of extracellular lipid correlates with pancreatic cancer subtype and metastatic tropism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C103.

Abstract A110: Clec4f is expressed in a subset of primary PDAC cells and confers invasive behavior

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly disease with a 5-year survival rate of just 12%. Poor survival is due in part to late detection of aggressive disease coincident with distant metastases, most often in the liver. While multiple proteins have been implicated in driving PDAC metastasis, effective targeted therapeutics have yet to be developed. Therefore, understanding factors contributing to PDAC liver metastasis has significant implications for diagnosis and treatment of the disease. Evidence has emerged to suggest that a subpopulation of cells with a pre-disposition for successful seeding and outgrowth in the liver are cultivated at the primary site prior to dissemination. Investigating public datasets generated from mouse models of PDAC liver metastasis, we noted upregulation of C-type Lectin Domain Family 4 Member F (Clec4f), a galactose-binding receptor. Importantly, we also detected Clec4f expression in a subset of primary PDAC tumor epithelial cells, which established our hypothesis that Clec4f is a novel regulator of PDAC dissemination. Preliminary in vitro invasion assays demonstrate that PDAC cells engineered to overexpress Clec4f exhibit more invasive behavior compared to control cells, displaying a more mesenchymal phenotype upon confluence in 2D culture and increased invasive protrusion when grown as 3D spheroids compared to control cells. To better examine tumor-intrinsic Clec4f-expressing PDAC cells in isolation, we sorted Clec4f-expressing cells from a mouse PDAC tumor via FACS to culture in vitro. However, upon establishment in culture, the sorted cells lost Clec4f expression. Importantly, Clec4f expression in other cell types has been shown to be dependent on niche-derived TGFβ signaling, which has also been shown to regulate tumor cell invasiveness and dissemination in PDAC. This led us to probe for Clec4f-expressing tumor cells in a mouse model of PDAC lacking TGFβ receptor 2 (PKT model). In the PKT model, we observed no Clec4f-expressing tumor cells, which suggested that niche-derived TGFβ signaling is required for the upregulation of Clec4f in PDAC. Using a variety of established cellular markers, we also determined that Clec4f was co-expressed in a subset of Synaptophysin (Syp) positive enteroendocrine cells in PDAC tissue. We are currently working to determine which niche components in the tumor microenvironment provide the necessary signals to upregulate Clec4f expression in PDAC tumor cells. We expect the outcome of this study to shed light upon pro-dissemination niche dynamics in PDAC and how they contribute to the deadly process of liver metastasis. Citation Format: Bailey A. Bye, Richard M. Walsh, Austin E. Eades, Michael N. VanSaun. Clec4f is expressed in a subset of primary PDAC cells and confers invasive behavior [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A110.

Anti-Prokineticin1 Suppresses Liver Metastatic Tumors in a Mouse Model of Colorectal Cancer with Liver Metastasis.

Multidisciplinary treatment for colorectal cancer (CRC) has undergone significant advances, and molecularly targeted drugs have substantially improved patient prognosis. However, one problem with current molecularly targeted therapeutics is that they must be used in combination with anticancer agents. New molecular targeted therapies that can be used alone are needed. We have previously identified prokineticin1 (PROK1) factor as a therapeutic potential target for CRC. PROK1 factor is involved in the angiogenesis of tissues surrounding CRC tumors. Additionally, PROK1 receptors 1 and 2 are expressed in CRC cell lines, playing roles in cell proliferation via an autocrine mechanism and in the signaling system. In this study, a liver metastasis mouse model was developed using human colorectal cancer cell lines, and mice were divided into anti-PROK1 antibody administration and control groups. Mice were treated intraperitoneally with antibodies or phosphate-buffered saline (control) every three days. The number, size, and cell proliferation ability of metastatic lesions were analyzed. Our results suggested that the number, size, and cancer cell proliferation ability of metastatic lesions decreased, and the survival time significantly increased in the antibody-treated group compared to those in the control group. Thus, the anti-PROK1 antibody therapy suppressed the cell proliferation ability of liver metastatic lesions in a CRC mouse model, suggesting its potential as a novel treatment strategy.

Modulating Gut Microbiota Prevents Anastomotic Leak to Reduce Local Implantation and Dissemination of Colorectal Cancer Cells after Surgery.

Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells. We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis. Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice. Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.

Discovery of a potent and highly selective inhibitor of SIRT6 against pancreatic cancer metastasis in vivo

Pancreatic cancer, one of the most aggressive malignancies, has no effective treatment due to the lack of targets and drugs related to tumour metastasis. SIRT6 can promote the migration of pancreatic cancer and could be a potential target for antimetastasis of pancreatic cancer. However, highly selective and potency SIRT6 inhibitor that can be used in vivo is yet to be discovered. Here, we developed a novel SIRT6 allosteric inhibitor, compound 11e, with maximal inhibitory potency and an IC50 value of 0.98 ± 0.13 μmol/L. Moreover, compound 11e exhibited significant selectivity against other histone deacetylases (HADC1‒11 and SIRT1‒3) at concentrations up to 100 μmol/L. The allosteric site and the molecular mechanism of inhibition were extensively elucidated by cocrystal complex structure and dynamic structural analyses. Importantly, we confirmed the antimetastatic function of such inhibitors in four pancreatic cancer cell lines as well as in two mouse models of pancreatic cancer liver metastasis. To our knowledge, this is the first study to reveal the in vivo effects of SIRT6 inhibitors on liver metastatic pancreatic cancer. It not only provides a promising lead compound for subsequent inhibitor development targeting SIRT6 but also provides a potential approach to address the challenge of metastasis in pancreatic cancer.

CHSY1 promotes CD8+ T cell exhaustion through activation of succinate metabolism pathway leading to colorectal cancer liver metastasis based on CRISPR/Cas9 screening

BackgroundThe most common site of metastasis in colorectal cancer (CRC) is the liver and liver metastases occur in more than 50% of patients during diagnosis or treatment. The occurrence of metastasis depends on a series of events known as the invasive-metastasis cascade. Currently, the underlying genes and pathways regulating metastasis initiation in the liver microenvironment are unknown.MethodsWe performed systematic CRISPR/Cas9 screening using an in vivo mouse model of CRC liver metastasis to identify key regulators of CRC metastasis. We present the full results of this screen,which included a list of genes that promote or repress CRC liver colonization. By silencing these genes individually, we found that chondroitin sulfate synthase 1 (CHSY1) may be involved in CRC metastasis. We verified the function of CHSY1 and its involvement in liver metastasis of CRC through in vivo and in vitro experiments.ResultThe results of TCGA and CRISPR/Cas9 showed that CHSY1 was overexpressed in CRC primary and liver metastasis tissues and indicated a worse clinical prognosis. In vitro and in vivo experiments confirmed that CHSY1 facilitated the liver metastasis of CRC and CHSY1 induced CD8+ T cell exhaustion and upregulated PD-L1 expression. The metabolomic analysis indicated that CHSY1 promoted CD8+ T cell exhaustion by activating the succinate metabolism pathway leading to liver metastasis of CRC. Artemisinin as a CHSY1 inhibitor reduced liver metastasis and enhanced the effect of anti-PD1 in CRC. PLGA-loaded Artemisinin and ICG probe reduced liver metastasis and increased the efficiency of anti-PD1 treatment in CRC.ConclusionCHSY1 could promote CD8+ T cell exhaustion through activation of the succinate metabolic and PI3K/AKT/HIF1A pathway, leading to CRC liver metastasis. The combination of CHSY1 knockdown and anti-PD1 contributes to synergistic resistance to CRC liver metastasis. Artemisinin significantly inhibits CHSY1 activity and in combination with anti-PD1 could synergistically treat CRC liver metastases. This study provides new targets and specific strategies for the treatment of CRC liver metastases, bringing new hope and benefits to patients.

Multifunctional nanodrug performs sonodynamic therapy and inhibits TGF-β to boost immune response against colorectal cancer and liver metastasis.

Liver metastasis is the leading cause of death in colorectal cancer. Immunotherapy using immune checkpoint blockade (ICB) is ineffective due to its immunological cold tumor nature. Herein, we prepared a nanodrug (NCG) encapsulating the transforming growth factor-β receptor inhibitor galunisertib (Gal) and the sonosensitizer chlorin e6 (Ce6), which was aimed to turn this type of cold tumor into a hot one to promote the ICB-based immunotherapy against it. After delivery to the tumor, NCG under ultrasonic irradiation generated reactive oxygen species causing tumor immunogenic cell death and releasing immunostimulatory signals such as calreticulin and HMGB1, which increased tumor immunogenicity and activated the innate T lymphocyte immune response. Moreover, NCG responded to the acidic microenvironment and released Gal, inhibiting phosphorylation and inducing immunosuppressive Smad2/3 signaling. Consequently, the differentiation of MDSCs was inhibited, M1-like polarization of tumor-associated macrophages was induced, and the immunosuppressive barrier of tumor-associated fibroblasts was destroyed to increase the infiltration of effector T cells, which reversed the immunosuppression of the tumor microenvironment and improved the therapeutic efficacy of anti-PD-L1 antibodies. Notably, in the liver metastasis mouse model, combination therapy using NCG (+) and aPD-L1 inhibited the growth of colon cancer liver metastasis, manifesting potential in treating this popular yet intractable malignancy. STATEMENT OF SIGNIFICANCE: Only a limited number of patients with colorectal cancer and liver metastasis can benefit from immune checkpoint blockade therapy, as most of them are microsatellite stable, immunologically cold tumors. Interestingly, there is compelling evidence that sonodynamic therapy (SDT) can convert immunosuppressed cold tumors into hot ones, trigger tumor immunogenic cell death non-invasively, and boost cytotoxic T cells infiltration. However, its therapeutic efficacy is constrained by the abundance of transforming growth factor-β (TGF-β) cytokines in the tumor microenvironment. Here, we reported a TGF-β-targeted inhibitory nanodrug that improved SDT in colon cancer and liver metastasis, reversed the immunosuppressive tumor microenvironment and boosted the immune response to anti-PD-L1 therapy in this cancer. It demonstrated the potential to cure this prevalent but incurable malignancy.

Colon-cancer liver metastasis is effectively targeted by recombinant methioninase (rMETase) in an orthotopic mouse model

BackgroundColorectal cancer liver metastasis (CCLM) is the most frequent cause of death of colorectal cancer. Development of novel new effective therapy is needed for CCLM patients to improve outcome. The aim of the present study was to investigate the efficacy of recombinant methioninase (rMETase) on a CCLM orthotopic mouse model of liver metastasis established using the human colon cancer cell line HT29 expressing red fluorescent protein (RFP). Materials and MethodsOrthotopic CCLM nude mouse models were randomized into two groups: control group (n = 6, PBS 200 µl, i.p., daily); rMETase group (n = 6, 100 units/200 µl, i.p., daily). Tumor volume was measured on day 0 and day 15. Body weight was measured twice a week. All mice were sacrificed on day 15. ResultsrMETase significantly inhibited the increase of the liver metastasis as determined by RFP fluorescence area and intensity (p = 0.016 and 0.015, respectively). There was no significant difference of body weight between either group on any day. ConclusionsThe present study suggests that rMETase has future potential therapy for CCLM in the clinic.

Emodin attenuates high lipid-induced liver metastasis through the AKT and ERK pathways in vitro in breast cancer cells and in a mouse xenograft model

Emodin, a natural anthraquinone derivative, can inhibit lipid synthesis and breast cancer cell proliferation. We previously found that emodin decreased breast cancer liver metastasis via epithelial-to-mesenchymal transition (EMT) inhibition. However, the mechanism through which emodin affects breast cancer liver metastasis in high-fat diet-induced obese and hyperlipidemic mice has not been elucidated. Bioinformatics analysis was used to reveal the potential targets and pathways of emodin. The mouse model of liver metastasis was established by injecting breast cancer cells into the left ventricle in high-fat diet-induced obese mice. The effect of emodin on inhibiting liver metastasis of breast cancer was evaluated by animal experiments. The mechanisms through which emodin inhibits liver metastasis of breast cancer were studied by cell and molecular biological methods. Emodin reduced lipid synthesis by inhibiting the expression of triglyceride (TG) synthesis-related genes, such as fatty acid synthase (Fasn), glycerol-3-phosphate acyltransferase 1 (Gpat1), and stearoyl-CoA desaturase (Scd1), and ultimately reduced liver metastasis in breast cancer. In addition, emodin inhibited breast cancer cell proliferation and invasion through the serine/threonine kinase (AKT) signaling and extracellular-regulated protein kinase (ERK) pathways by interacting with CSNK2A1, ESR1, ESR2, PIM1 and PTP4A3. Our results indicate that emodin may have therapeutic potential in the prevention or treatment of breast cancer liver metastasis.

The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells.

Gastric cancer (GC) is a malignant tumour with high mortality, and liver metastasis is one of the main causes of poor prognosis. SLIT- and NTRK-like family member 4 (SLITRK4) plays an important role in the nervous system, such as synapse formation. Our study aimed to explore the functional role of SLITRK4 in GC and liver metastasis. The mRNA level of SLITRK4 was evaluated using publicly available transcriptome GEO datasets and Renji cohort. The protein level of SLITRK4 in the tissue microarray of GC was observed using immunohistochemistry. Cell Counting Kit-8, colony formation, transwell migration assays in vitro and mouse model of liver metastasis in vivo was performed to investigate the functional roles of SLITRK4 in GC. Bioinformatics predictions and Co-IP experiments were applied to screen and identify SLITRK4-binding proteins. Western blot was performed to detect Tyrosine Kinase receptor B (TrkB)-related signaling molecules. By comparing primary and liver metastases from GC, SLITRK4 was found to be upregulated in tissues of GC with liver metastasis and to be closely related topoor clinical prognosis. SLITRK4 knockdown significantly abrogated the growth, invasion, and metastasis of GC in vitro and in vivo. Further study revealed that SLITRK4 could interact with Canopy FGF Signalling Regulator 3 (CNPY3), thus enhancing TrkB- related signaling by promoting the endocytosis and recycling of the TrkB receptor. In conclusion, the CNPY3-SLITRK4 axis contributes to liver metastasis of GC according to the TrkB-related signaling pathway. which may be a therapeutic target for the treatment of GC with liver metastasis.

Macrophage STING signaling promotes NK cell to suppress colorectal cancer liver metastasis via 4-1BBL/4-1BB co-stimulation

Background and aimsMacrophage innate immune response plays an important role in tumorigenesis. However, the role and mechanism of macrophage STING signaling in modulating tumor microenvironment to suppress tumor growth at...

Dynamic differentiation of F4/80+ tumor-associated macrophage and its role in tumor vascularization in a syngeneic mouse model of colorectal liver metastasis

Tumor-associated macrophages (TAMs) are highly heterogeneous and play vital roles in tumor progression. Here we adopted a C57BL/6 mouse model imitating the late-stage colorectal liver metastasis (CRLM) by Mc38 colorectal cancer cell injection via the portal vein. With serial sections of CRLM biopsies, we defined 7–9 days post-injection as the critical period for tumor neovascularization, which was initiated from the innate liver vessels via vessel cooption and extended by vascular mimicry and thereof growth of CD34+cells. In samples with increasing-sized liver metastases, the infiltrated Ly6C+ CD11b+ F4/80− monocytes steadily gained the expression of F4/80, a Kupffer cell marker, before transformed into Ly6C− CD11bint F4/80+ cells, which, the same phenotype was also adapted by Ly6C− CD11b− F4/80+ Kupffer cells. F4/80+ TAMs showed proximity to neovascularization and tumor vessels, functionally angiogenic in vivo; and greatly promoted the activation of a few key angiogenic markers such as VEGFA, Ki67, etc. in endothelial cells in vitro. Depletion of macrophages or diversion of macrophage polarization during neovascularization impeded tumor growth and vascularization and resulted in greatly reduced F4/80+ TAMs, yet increased CD11b+ cells due to inhibition of TAM differentiation. In summary, our results showed dynamic and spatial–temporal F4/80+ TAM transformation within the tumor microenvironment and strengthened its role as perivascular and angiogenic TAMs in CRLM.

A reliable mouse model of liver and lung metastasis by injecting esophageal cancer stem cells (CSCs) through tail-vein injection.

Esophageal Squamous Cell Carcinoma (ESCC) is a highly aggressive tumor with increased metastatic potential. Recent evidence suggests that esophageal CSCs have a crucial role in tumor initiation, progression, and resistance to conventional anti-cancer therapies. The study aimed to develop mouse model to mimic the late steps of the metastasis process using a tail-vein injection of esophageal CSCs. The sphere formation assay was used to enrich CSCs. For analysis of tumorigenicity, YM-1 adherent cells and enriched CSCs were injected subcutaneously into dorsal flank of nude mice. The expression of SLUG, E-cad, and CTHRC1 genes was examined by Real-Time qRT-PCR and immunohistochemistry (IHC) methods. To assess the metastatic potential of adherent YM-1 cells and their enriched CSCs, we injected the cells into the tail vein of nude mice. Our findings showed the up-regulation of SLUG and down-regulation of E-cad in the esophageal CSC-derived tumors (ECSCTs) compared to adherent cells-derived tumors. There was no statistically significant difference between CTHRC1 gene expressions in both groups of tumors. IHC staining confirmed the higher expression of SLUG protein in ECSCTs compared to adherent cell-derived tumors. Enriched CSCs were able to metastasize to the lungs and livers after three months, but, metastasis of adherent cells wasn't observed. Our study showed esophageal CSCs injected through the tail-vein injection can migrate and metastasize to the lung and liver after three months. The developed metastatic mouse model can be a valuable and relevant model to investigate the molecular and cellular mechanisms of metastasis and develop successful targeted therapies against ESCC. The present study is one of the few studies that investigate the metastasis of esophageal cancer stem cells (ESCC type) through injection into the tail vein of nude mice.

The anti-metastasis effect of low-dose carbon monoxide

: Metastasis is the primary cause of cancer-related deaths. Because of the anatomically diffused localizations in different organs, treating metastatic cancer with conventional surgery or radiotherapy is challenging. Systemic therapies, including chemotherapy, immunotherapy, and targeted therapy, are used for metastatic cancer treatment. However, resistance to chemotherapy often develops over time, and immunotherapy or targeted therapy is not available for every cancer type. Improved systemic therapies for metastatic cancer are urgently needed. Among all forms of cancer, pancreatic ductal adenocarcinoma (PDAC) is a lethal type due to the following characteristics: early distant metastasis, abundant desmoplastic stroma, insufficient therapeutics, and frequent chemoresistance. Carbon monoxide (CO) is produced endogenously in humans and has been established as an important, biologically active signaling molecule. Accumulating evidence suggests the therapeutic applications of safe, low-dose CO for various diseases. Recently, low-dose CO was shown to suppress metastasis of PDAC in an orthotopic mouse model of liver metastasis and a spontaneous PDAC metastasis mouse model after the removal of primary tumor xenograft tumors. In this mini-review, we discuss the findings and the potential development of CO-based systemic therapies for metastatic cancer. Specifically, many non-inhalation CO delivery modalities with easy control of dosages have been developed and can be further studied for their anti-metastatic properties.

Tumor-associated macrophages mediate gastrointestinal stromal tumor cell metastasis through CXCL2/CXCR2.

Tumor-associated macrophages (TAMs) are linked with the progression and poor prognosis of multifarious solid tumors, but the regulatory mechanisms involved in gastrointestinal stromal tumors (GIST) remain indistinct. This study intended to delve into the job of TAM-derived chemokines in promoting metastasis in GIST microenvironment. Expression levels of M2-TAM markers and CXCL2 in primary and metastatic tissues of GIST were analyzed by bioinformatics methods, and we analyzed the correlation between CXCL2 and M2-TAM markers. Immunofluorescence was applied to assay CXCL2 and M2-TAM marker protein (CD68 and CD206) expression in tumor tissues. Serum CXCL2 concentration in metastatic and non-metastatic patients was assayed by ELISA. The differentiation of THP-1 cells was tested by flow cytometry. Cell function test was utilized to analyze the viability, invasion and migration of GIST cells. Western blot was used to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins. The mouse liver metastasis model was established, and the effects of CXCL2 and EMT-related genes on metastasis were confirmed by hematoxylin-eosin staining and immunohistochemistry experiments. Bioinformatics analysis ascertained that M2-TAM marker proteins and chemokine CXCL2 were highly expressed in GIST metastatic tissues, and CXCL2 and TAM were co-located in tumor tissues. Results of in vitro cell function experiments displayed that CXCL2 secreted by M2-TAM promoted the invasion, migration and EMT of GIST tumor cells, and the anti-CXCL2 antibody could block the metastasis promoting effect of CXCL2. Additionally, the silencing of CXCR2 in GIST cells inhibited the metastasis promoting effect of CXCL2. Animal studies further confirmed that CXCL2 promoted liver metastasis of GIST in vivo. This study preliminarily revealed the mechanism of M2-TAM promoting tumor metastasis by secreting CXCL2 in GIST tumor microenvironment, and proffered theoretical reference for the development of immunotherapy strategies targeting M2-TAM.