Formation Of Liver Metastases Research Articles

A Paradoxical Tumor Antigen Specific Response in the Liver.

Functional tumor-specific CD8+ T cells are essential for an effective anti-tumor immune response and the efficacy of immune checkpoint inhibitor therapy. In comparison to other organ sites, we found higher numbers of tumor-specific CD8+ T cells in primary, metastatic liver tumors in murine tumor models. Despite their abundance, CD8+ T cells in the liver displayed an exhausted phenotype. Depletion of CD8+ T cells showed that liver tumor-reactive CD8+ T failed to control liver tumors but was effective against subcutaneous tumors. Similarly, analysis of single-cell RNA sequencing data from patients showed a higher frequency of exhausted tumor-reactive CD8+ T cells in liver metastasis compared to paired primary colon cancer. High-dimensional, multi-omic analysis combining proteomic CODEX and scRNA-seq data revealed enriched interaction of SPP1+ macrophages and CD8+ tumor-reactive T cells in profibrotic, alpha-SMA rich regions in the liver. Liver tumors grew less in Spp1-/- mice and the tumor-specific CD8+ T cells were less exhausted. Differential pseudotime trajectory inference analysis revealed extrahepatic signaling promoting an intermediate cell (IC) population in the liver, characterized by co-expression of VISG4, CSF1R, CD163, TGF-βR, IL-6R, SPP1. scRNA-seq of a third data set of premetastatic adenocarcinoma showed that enrichment of this population may predict liver metastasis. Our data suggests a mechanism by which extrahepatic tumors facilitate the formation of liver metastasis by promoting an IC population inhibiting tumor-reactive CD8+ T cell function.

Targeting circ-0034880-enriched tumor extracellular vesicles to impede SPP1highCD206+ pro-tumor macrophages mediated pre-metastatic niche formation in colorectal cancer liver metastasis

BackgroundInformation transmission between primary tumor cells and immunocytes or stromal cells in distal organs is a critical factor in the formation of pre-metastatic niche (PMN). Understanding this mechanism is essential for developing effective therapeutic strategy against tumor metastasis. Our study aims to prove the hypothesis that circ-0034880-enriched tumor-derived extracellular vesicles (TEVs) mediate the formation of PMN and colorectal cancer liver metastasis (CRLM), and targeting circ-0034880-enriched TEVs might be an effective therapeutic strategy against PMN formation and CRLM.MethodsWe utilized qPCR and FISH to measure circRNAs expression levels in human CRC plasma, primary CRC tissues, and liver metastatic tissues. Additionally, we employed immunofluorescence, RNA sequencing, and in vivo experiments to assess the effect mechanism of circ-0034880-enriched TEVs on PMN formation and CRC metastasis. DARTS, CETSA and computational docking modeling were applied to explore the pharmacological effects of Ginsenoside Rb1 in impeding PMN formation.ResultsWe found that circ-0034880 was highly enriched in plasma extracellular vesicles (EVs) derived from CRC patients and closely associated with CRLM. Functionally, circ-0034880-enriched TEVs entered the liver tissues and were absorbed by macrophages in the liver through bloodstream. Mechanically, TEVs-released circ-0034880 enhanced the activation of SPP1highCD206+ pro-tumor macrophages, reshaping the metastasis-supportive host stromal microenvironment and promoting overt metastasis. Importantly, our mechanistic findings led us to discover that the natural product Ginsenoside Rb1 impeded the activation of SPP1highCD206+ pro-tumor macrophages by reducing circ-0034880 biogenesis, thereby suppressing PMN formation and inhibiting CRLM.ConclusionsCirc-0034880-enriched TEVs facilitate strong interaction between primary tumor cells and SPP1highCD206+ pro-tumor macrophages, promoting PMN formation and CRLM. These findings suggest the potential of using Ginsenoside Rb1 as an alternative therapeutic agent to reshape PMN formation and prevent CRLM.

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Blocking the angiopoietin-2-dependent integrin β-1 signaling axis abrogates small cell lung cancer invasion and metastasis.

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin β-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin β-1 or blocking Integrin β-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin β-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin β-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

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.

Cancer-Derived Small Extracellular Vesicles Affect Vascular Endothelial Cells and Promote Adhesiveness of Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the most aggressive malignancies due to the high rate of metastasis. The mechanisms underlying metastasis need to be elucidated. Small extracellular vesicles (sEVs) mediate cell-to-cell communication, and cancer-derived sEVs contribute to the formation of premetastatic niches. The present study examined changes in adhesiveness by the internalization of PC-derived sEVs into vascular endothelial cells, and investigated the molecular mechanisms underlying metastasis. Pancreatic cancer-derived sEVs were internalized into vascular endothelial cells, and changes in adhesiveness were evaluated. We evaluated the effects of sEVs on the formation of liver metastasis in vivo. We also assessed molecular changes in vascular endothelial cells by the internalization of PC-derived sEVs. The internalization of PC-derived sEVs into vascular endothelial cells promoted the adhesiveness of vascular endothelial cells and PC cells. Pancreatic cancer-derived sEVs contained high levels of transforming growth factor β1 mRNA and acted as its transporter. Once PC-derived sEVs were internalized into vascular endothelial cells, the expression of fibronectin 1 increased on the cell surface, and the adhesiveness of vascular endothelial cells was enhanced. We investigated association between PC-derived sEVs and adhesiveness. Regulation of PC-derived sEVs has potential as a therapeutic modality to suppress the metastasis of PC.

KLK10 derived from tumor endothelial cells accelerates colon cancer cell proliferation and hematogenous liver metastasis formation.

Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation invivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation invivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.

Abstract A041: 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 particularly poor prognosis. Targeted 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 first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. 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. 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 performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generate a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched in both. Next, we validated genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determined 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 with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified SerpinE2, a protease inhibitor, as a target that could drive breast cancer metastasis and cell competition. 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 (WT) 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 WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively 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. Citation Format: Katherine E Lake, Sakshi Mohta, Clayton A Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Jacob Pena, Luise Chinea, Luisa Froessl, Sangeetha M Reddy, Heather L McArthur, Christine Hodges, Julia Maues, Elizabeth Chen, Isaac S Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A041.

Natural killer cells drive 4-1BBL positive uveal melanoma towards EMT and metastatic disease

BackgroundInflammation in the eye is often associated with aggravated ocular diseases such as uveal melanoma (UM). Poor prognosis of UM is generally associated with high potential of metastatic liver dissemination. A strong driver of metastatic dissemination is the activation of the epithelial-mesenchymal transition (EMT) regulating transcription factor ZEB1, and high expression of ZEB1 is associated with aggressiveness of UM. While ZEB1 expression can be also associated with immune tolerance, the underlying drivers of ZEB1 activation remain unclear.MethodsTranscriptomic, in vitro, ex vivo, and in vivo analyses were used to investigate the impact on clinical prognosis of immune infiltration in the ocular tumor microenvironment. A metastatic liver dissemination model of was developed to address the role of natural killer (NK) cells in driving the migration of UM.ResultsIn a pan-cancer TCGA analysis, natural killer (NK) cells were associated with worse overall survival in uveal melanoma and more abundant in high-risk monosomy 3 tumors. Furthermore, uveal melanoma expressed high levels of the tumor necrosis factor superfamily member 4-1BB ligand, particularly in tumors with monosomy 3 and BAP1 mutations. Tumors expressing 4-1BB ligand induced CD73 expression on NK cells accompanied with the ability to promote tumor dissemination. Through ligation of 4-1BB, NK cells induced the expression of the ZEB1 transcription factor, leading to the formation of liver metastasis of uveal melanoma.ConclusionsTaken together, the present study demonstrates a role of NK cells in the aggravation of uveal melanoma towards metastatic disease.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy invivo. To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

CD4+ T cell-derived IL-22 enhances liver metastasis by promoting angiogenesis

ABSTRACT Metastasis is a cancer-related systemic disease and is responsible for the greatest mortality rate among cancer patients. Interestingly, the interaction between the immune system and cancer cells seems to play a key role in metastasis formation in the target organ. However, this complex network is only partially understood. We previously found that IL-22 produced by tissue resident iNKT17 cells promotes cancer cell extravasation, the early step of metastasis. Based on these data, we aimed here to decipher the role of IL-22 in the last step of metastasis formation. We found that IL-22 levels were increased in established metastatic sites in both human and mouse. We also found that Th22 cells were the key source of IL-22 in established metastasis sites, and that deletion of IL-22 in CD4+ T cells was protective in liver metastasis formation. Accordingly, the administration of a murine IL-22 neutralizing antibody in the establishment of metastasis formation significantly reduced the metastatic burden in a mouse model. Mechanistically, IL-22-producing Th22 cells promoted angiogenesis in established metastasis sites. In conclusion, our findings highlight that IL-22 is equally as important in contributing to metastasis formation at late metastatic stages, and thus, identify it as a novel therapeutic target in established metastasis.

IL-22BP controls the progression of liver metastasis in colorectal cancer.

The immune system plays a pivotal role in cancer progression. Interleukin 22 binding protein (IL-22BP), a natural antagonist of the cytokine interleukin 22 (IL-22) has been shown to control the progression of colorectal cancer (CRC). However, the role of IL-22BP in the process of metastasis formation remains unknown. We used two different murine in vivo metastasis models using the MC38 and LLC cancer cell lines and studied lung and liver metastasis formation after intracaecal or intrasplenic injection of cancer cells. Furthermore, IL22BP expression was measured in a clinical cohort of CRC patients and correlated with metastatic tumor stages. Our data indicate that low levels of IL-22BP are associated with advanced (metastatic) tumor stages in colorectal cancer. Using two different murine in vivo models we show that IL-22BP indeed controls the progression of liver but not lung metastasis in mice. We here demonstrate a crucial role of IL-22BP in controlling metastasis progression. Thus, IL-22 might represent a future therapeutic target against the progression of metastatic CRC.

Colorectal cancer-derived small extracellular vesicles induce TGFβ1-mediated epithelial to mesenchymal transition of hepatocytes

BackgroundMetastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called “pre-metastatic niche”. In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown.MethodssEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment.ResultsOur study shows for the first time that TGFβ1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFβ1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients.ConclusionsSince it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Abstract 2484: The role of the PI3K/Akt signaling pathway on LIN28B-mediated colorectal cancer metastasis

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death worldwide. The 5-year survival rate is 90% in patients with localized tumors, but the survival rate drastically decreases to 14% in patients with distant metastasis. Therefore, it is imperative to elucidate the molecular mechanisms underlying CRC metastasis. The RNA-binding protein LIN28B is overexpressed in over 30% of patients with CRC and is associated with poor prognosis. Indeed, our previous work revealed that LIN28B promotes colorectal polyps and tumors in a genetic mouse model (PMID: 24142874) and metastasis in a subcutaneous xenograft model of CRC (PMID: 21512136). In the present study, we investigated the mechanism by which LIN28B promotes colorectal tumor progression and metastasis. To assess LIN28B-mediated biological changes, we established CRC cells (DLD-1 and LoVo) with overexpression of LIN28B. LIN28B overexpression increased the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in CRC cells. Knockdown of claudin-1 (CLDN1) or NOTCH3 expression in LIN28B-overexpresed CRC cells, or treatment of the cells with the pan-Notch inhibitor DAPT (γ-secretase inhibitor), revealed that the PI3K/Akt pathway works downstream of the tight junction protein CLDN1 and the transmembrane receptor NOTCH3. Furthermore, cell-cell contact of CRC cells was required for the activation of the NOTCH3 and PI3K/Akt signaling pathways, suggesting for an important role of CLDN1 as a tight junction protein upstream of NOTCH3 and PI3K. Inhibition of PI3K by either the pan-PI3K inhibitor LY294002 or PI3K-α-specific inhibitor Alpelisib (BYL719) suppressed LIN28B-mediated CRC cell migration and wound healing rate. Using a mouse model of metastatic CRC, we show that Alpelisib treatment significantly reduced LIN28B-induced liver metastases formation. Interestingly, the inhibitory effect of Alpelisib was even more pronounced when Alpelisib was given in combination with the pan-Notch inhibitor DAPT, resulting in complete inhibition of visible liver metastases formation. Immunohistochemical staining revealed that Alpelisib did not affect proliferation, apoptosis, or angiogenesis of the liver metastases, whereas DAPT may inhibit angiogenesis. Taken together, our results indicate that LIN28B promotes cell migration and liver metastasis formation through CLDN1 and NOTCH3-mediated activation of PI3K signaling. Development of new therapies that target the PI3K/Akt pathway may provide an effective strategy for preventing metastases in CRC patients. This work is supported by NIH 9R01CA277795-22, NIH 5P30CA0113696 and the American Cancer Society. The authors declare no conflicts of interest. Citation Format: Alice E. Shin, Kensuke Sugiura, Kensuke Suzuki, Yasunori Masuike, Christopher J. Lengner, Anil K. Rustgi. The role of the PI3K/Akt signaling pathway on LIN28B-mediated colorectal cancer 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 2484.

Abstract 3490: IGFBP1 mediates musculoskeletal defects in colorectal cancer

Abstract Colorectal cancer (CRC) is frequently accompanied by cachexia, an uncured multi-systemic wasting syndrome that affects the majority of patients, especially when the disease recurs with liver metastases (LMs). Muscle and bone loss are amongst the most detrimental symptoms of cachexia and directly cause increased morbidity and mortality. We and others have shown that CRC also promotes metabolic and genomic perturbations of the liver, and further, that formation of LMs exacerbates muscle and bone wasting. These observations, along with evidence that liver-derived factors (i.e., hepatokines) may poorly influence musculoskeletal health, suggest an endocrine role of the liver in mediating cancer-induced cachexia. In the present study, we identified the hepatokine insulin-like growth factor binding protein 1 (IGFBP1) as a novel mediator of musculoskeletal wasting in CRC. Plasma from CRC patients and preclinical mouse models of CRC (C26, MC38, HCT116, ApcMin/+) were assessed for circulating IGFBP1 levels. AML12 hepatocytes were cultured with CRC cells (C26, MC38, HCT116) to assess tumor induced hepatic IGFBP1 production, while C2C12 myotubes and osteoclast precursor cells were cultured to examine the in vitro effects of IGFBP1 on myotube atrophy and osteoclastogenesis. 8-week-old male wild-type (WT) C57BL/6J and IGFBP1-KO mice were intrasplenically injected with MC38 tumor cells (mMC38) to mimic hepatic dissemination of cancer cells, while sham-operated animals received saline. Animals were assessed for muscle force 24-hours prior to euthanasia, and skeletal muscles and bone were collected for molecular and morphological analyses. CRC patients and CRC tumor-bearing mice demonstrated markedly elevated circulating plasma IGFBP1, also supported by increased liver IGFBP1 mRNA expression in C26, MC38 and HCT116 hosts. Follow-up in vitro studies demonstrated that co-culturing CRC cells (lacking IGFBP1 expression) with AML12 hepatocytes promotes IGFBP1 production, thereby suggesting that IGFBP1 is purely host-derived. Further, treatment with recombinant IGFBP1 was sufficient to stimulate osteoclastogenesis, while also promoting atrophy of C2C12 myotubes. Conversely, use of anti-IGFBP1 neutralizing antibodies prevented osteoclastogenesis and preserved C2C12 myotube size when exposed to plasma from mice bearing CRCs. Notably, WT mMC38 bearers displayed reductions in muscle mass and strength, as well as in trabecular bone volume fraction (BV/TV) and trabecular number (Tb.N). Conversely, IGFBP1-KO tumor hosts exhibited preserved skeletal muscle and bone mass. Altogether, our data implicate IGFBP1, an exquisitely liver-derived factor, as a novel mediator of musculoskeletal deficits in CRC cachexia, and supports novel strategies to counteract host-derived factors in the treatment of cancer-associated multi-organ complications. Citation Format: Joshua R. Huot, Fabrizio Pin, Andrea Bonetto. IGFBP1 mediates musculoskeletal defects in 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 3490.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer

Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, invitro, and invivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

Tumor-associated macrophage-derived GDNF promotes gastric cancer liver metastasis via a GFRA1-modulated autophagy flux

PurposeLiver metastasis, a lethal malignancy of gastric cancer (GC) patients, execrably impairs their prognosis. As yet, however, few studies have been designed to identify the driving molecules during its formation, except screening evidence pausing before their functions or mechanisms. Here, we aimed to survey a key driving event within the invasive margin of liver metastases.MethodsA metastatic GC tissue microarray was used for exploring malignant events during liver-metastasis formation, followed by assessing the expression patterns of glial cell-derived neurotrophic factor (GDNF) and GDNF family receptor alpha 1 (GFRA1). Their oncogenic functions were determined by both loss- and gain-of-function studies in vitro and in vivo, and validated by rescue experiments. Multiple cell biological studies were performed to identify the underlying mechanisms.ResultsIn the invasive margin, GFRA1 was identified as a pivotal molecule involved in cellular survival during liver metastasis formation, and we found that its oncogenic role depends on tumor associated macrophage (TAM)-derived GDNF. In addition, we found that the GDNF-GFRA1 axis protects tumor cells from apoptosis under metabolic stress via regulating lysosomal functions and autophagy flux, and participates in the regulation of cytosolic calcium ion signalling in a RET-independent and non-canonical way.ConclusionFrom our data we conclude that TAMs, homing around metastatic nests, induce the autophagy flux of GC cells and promote the development of liver metastasis via GDNF-GFRA1 signalling. This is expected to improve the comprehension of metastatic pathogenesis and to provide a novel direction of research and translational strategies for the treatment of metastatic GC patients.

Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Lyve-1 deficiency enhances the hepatic immune microenvironment entailing altered susceptibility to melanoma liver metastasis.

Hyaluronan receptor LYVE-1 is expressed by liver sinusoidal endothelial cells (LSEC), lymphatic endothelial cells and specialized macrophages. Besides binding to hyaluronan, LYVE-1 can mediate adhesion of leukocytes and cancer cells to endothelial cells. Here, we assessed the impact of LYVE-1 on physiological liver functions and metastasis. Mice with deficiency of Lyve-1 (Lyve-1-KO) were analyzed using histology, immunofluorescence, microarray analysis, plasma proteomics and flow cytometry. Liver metastasis was studied by intrasplenic/intravenous injection of melanoma (B16F10 luc2, WT31) or colorectal carcinoma (MC38). Hepatic architecture, liver size, endothelial differentiation and angiocrine functions were unaltered in Lyve-1-KO. Hyaluronan plasma levels were significantly increased in Lyve-1-KO. Besides, plasma proteomics revealed increased carbonic anhydrase-2 and decreased FXIIIA. Furthermore, gene expression analysis of LSEC indicated regulation of immunological pathways. Therefore, liver metastasis of highly and weakly immunogenic tumors, i.e. melanoma and colorectal carcinoma (CRC), was analyzed. Hepatic metastasis of B16F10luc2and WT31 melanoma cells, but not MC38 CRC cells, was significantly reduced inLyve-1-KO mice. In vivo retention assays with B16F10luc2cells were unaltered between Lyve-1-KO and control mice. However, in tumor-free Lyve-1-KO livers numbers of hepatic CD4+, CD8+ and regulatory Tcells were increased. In addition, iron deposition was found in F4/80+ liver macrophages known to exert pro-inflammatory effects. Lyve-1 deficiency controlled hepatic metastasis in a tumor cell-specific manner leading to reduced growth of hepatic metastases of melanoma, but not CRC. Anti-tumorigenic effects are likely due to enhancement of the premetastatic hepatic immune microenvironment influencing early liver metastasis formation.

Emerging mechanisms progress of colorectal cancer liver metastasis.

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. A total of 20% of CRC patients present with distant metastasis. The hepatic portal venous system, responsible for collecting most intestinal blood, makes the liver the most common site of CRC metastasis. The formation of liver metastases from colorectal cancer is a long and complex process. It involves the maintenance of primary tumors, vasculature invasion, distant colonization, and metastasis formation. In this review, we serve on how the CRC cells acquire stemness, invade the vascular, and colonize the liver. In addition, we highlight how the resident cells of the liver and immune cells interact with CRC cells. We also discuss the current immunotherapy approaches and challenges we face, and finally, we look forward to finding new therapeutic targets based on novel sequencing technologies.