Experimental Metastasis Model Research Articles

EDI3 knockdown in ER-HER2+ breast cancer cells reduces tumor burden and improves survival in two mouse models of experimental metastasis

BackgroundDespite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis.MethodsTo inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively.ResultsInducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo.ConclusionsReduced metastasis upon silencing supports EDI3’s potential as a treatment target in metastasizing ER-HER2+ breast cancer.

Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines

BackgroundCirculating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters.MethodsThe assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used.ResultsWe systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC.ConclusionsThe study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

Abstract 1495: Lipocalin-2 promotes breast cancer metastasis via tumor cell intrinsic and extrinsic mechanisms

Abstract While metastasis is the primary factor predicting poor outcomes in solid tumors, survival is lowest in patients initially diagnosed with a localized malignancy that then progresses to metastatic disease. Notably, nearly 25% of early-stage breast cancers (BCs) metastasize during treatment or following an initial positive response to therapeutic intervention. To define the molecular and cellular factors that contribute to metastatic progression of BC, we previously developed the Py230-C57Bl/6 syngeneic experimental metastasis model for interrogating mechanisms by which soluble factors condition premetastatic lung tissue to potentiate BC cell seeding/expansion. Using this model and the TCGA, we identified lipocalin-2 (Lcn2) as a molecular candidate that is upregulated in lung-tropic Py230 cells and other human triple-negative breast cancer (TNBC) cells, and that predicts poor outcomes in human BCs. While Lcn2 has been previously reported to mediate BC progression in genetically engineered mouse models, its mechanism(s) of action remain undefined. Here, we demonstrate that Py230 cell-secreted Lcn2 is necessary for optimal reprogramming of the premetastatic lung during metastatic progression. In agreement with these data, we defined a cell intrinsic role for Lcn2 in BC cell organoid assembly/invasion and expression of proinflammatory factors. To define BC cell extrinsic roles for Lcn2 in remodeling the premetastatic lung, we collected global transcriptome and single-cell spatial proteome data for lung tissue from C57Bl/6 mice systemically educated with mock media or media conditioned with Py8119 (Lcn2low) or Py230 (Lcn2hi) BC cells. Mice receiving Py230 conditioned media treatments were further divided between two groups – one receiving an isotype control IgG and the other receiving an Lcn2 immunoneutralizing antibody. Global transcriptome data was used to determine novel gene set enrichment signatures and clinically relevant interactomes – notable candidates predicted to cooperate with Lcn2 in decreasing patient survival were affiliated with cilia biogenesis and function. Single-cell spatial proteome data were analyzed for 28 different cell populations within the premetastatic lung. Interestingly, Py230 cell-derived Lcn2 did not affect the percentage of ciliated epithelial cells indicating that the global transcriptome data referenced above represent a real increase in expression of cilia-related genes and not an increase in cilia-positive cell types. Notably, we identified that Lcn2 increased the percentage of CD45neg/Despos mechanomodulatory cells and proliferating CD45pos/CD3pos/CD4neg/CD8pos cytotoxic T cells in the premetastatic lung. Taken together, these data define new targets around which new therapeutic strategies can be developed to improve BC patient outcomes. Citation Format: Joshua Gamez, Francesca Sanchez, Gabriela Ortiz-Soto, Jane Cox, Amir Gerami, Jonathan A. Kelber. Lipocalin-2 promotes breast cancer metastasis via tumor cell intrinsic and extrinsic mechanisms [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 1495.

Platelets induce CD39 expression in tumor cells to facilitate tumor metastasis.

Tumor cells continue to evolve the metastatic potential in response to signals provided by the external microenvironment during metastasis. Platelets closely interact with tumor cells during hematogenous metastasis and facilitate tumor development. However, the molecular mechanisms underlying this process are not fully understood. RNA-sequencing was performed to screen differentially expressed genes mediated by platelets. The effects of platelet and CD39 on tumor metastasis were determined by experimental metastasis models with WT, NCG and CD39-/- mice. RNA-sequencing results showed that platelets significantly up-regulated CD39 expression in tumor cells. CD39 is a novel immune checkpoint molecule and a key driver of immunosuppression. Our data provided evidence that the expression of CD39 was enhanced by platelets in a platelet-tumor cell contact dependent manner. Although the role of CD39 expressed by immune cells is well established, the effect of CD39 expressed by tumor cells on tumor cell behavior, anti-tumor immunity and tumor metastasis is unclear. We found that CD39 promoted tumor cell invasion, but had no effect on proliferation and migration. Notably, we showed that the ability of platelets to prime tumor cells for metastasis depends on CD39 in the experimental tumor metastasis model. CD39 silencing resulted in fewer experimental metastasis formation, and this anti-metastasis effect was significantly reduced in platelet-depleted mice. Furthermore, overexpression of CD39 in tumor cells promoted metastasis. In order to eliminate the effect of CD39 expressed in cells other than tumor cells, we detected tumor metastasis in CD39-/- mice and obtained similar results. Moreover, overexpression of CD39 in tumor cells inhibited antitumor immunity. Finally, the data from human samples also supported our findings. Our study shows that direct contact with platelets induces CD39 expression in tumor cells, leading to immune suppression and promotion of metastasis.

Abstract B103: The impact of ULK1 deficiency on autophagy, cell viability, and tumor progression in experimental models of ovarian cancer metastasis

Abstract B103: The impact of ULK1 deficiency on autophagy, cell viability, and tumor progression in experimental models of ovarian cancer metastasis

Abstract A018: Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival and metastasis of HER2-positive and basal breast cancer through regulation of caspase 14

Abstract Despite improvements in therapies, metastasis still leads to high mortality rates in breast cancer (BC). Consequently, discovering new therapies that are effective at targeting metastatic disease remains a critical need, particularly for the aggressive HER2+ and basal BC subtypes. The ability of cancer cells to survive in the absence of an extracellular matrix – termed anchorage-independent survival (AIS) – is central to the metastatic cascade and has emerged as a target biology of interest for reducing metastatic burden. Previously, we identified the SL enzyme dihydroceramide desaturase 1 (DES1) as a critical node for AIS of HER2+ BC cells. Here, we have extended these data to show that targeting DES1 through pharmacological inhibitors (4HPR), siRNA, or CRISPR-cas9 knockout was able to overcome AIS in basal BC cells as well as HER2+ BC cells that, functionally, led to a reduction of in vitro colony formation in soft agar. Extending these findings in vivo, we found that DES1 knockout led to a significant reduction in lung metastasis of both HER2+ and basal BC cells in murine models of experimental metastasis. Moreover, treatment of PyMT mice with the DES1 inhibitor 4HPR was able to reduce metastatic burden. To define downstream effectors of DES1 required for AIS, we performed a microarray analysis of control and DES1 knockout cells in suspension, leading us to identify caspase 14 as a downstream effector of DES1 in HER2+ and basal BC. Consistent with these findings, siRNA knockdown of caspase 14 led to a striking decrease in cell viability in colony formation assays. Finally, analysis of publicly available data found that high levels of DES1 and Caspase 14 were associated with poor clinical outcomes in both HER2+ and basal BC. Taken together, these data establish DES1 as a mediator of BC metastasis, likely through promoting AIS, and define caspase 14 as a target of DES1-driven signaling. Furthermore, results suggest that both DES1 and caspase 14 expression may be useful as biomarkers of metastasis-prone tumors. Citation Format: Deanna M Peperno, Danielle Lambadis, Victoria Alvarado, Andrew E Resnick, Christopher J Clarke. Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival and metastasis of HER2-positive and basal breast cancer through regulation of caspase 14 [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 A018.

Unique macrophage phenotypes activated by BMP signaling in breast cancer bone metastases.

Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow-derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.

Resveratrol-derived inhibitors of the E3 ubiquitin ligase PELI1 inhibit the metastasis of triple-negative breast cancer

Triple-negative breast cancer (TNBC), as the most challenging subtype of breast cancer, exerts highly invasive ability and metastatic nature to the lymph nodes, which is correlated with poor survival rates among patients. Pellino-1 (PELI1) is an E3 ubiquitin ligase involved in tumor invasion and metastasis, and has the potential to be developed as a novel therapeutic target for TNBC. In this study, we identified a potent inhibitor of PELI1, namely compound 3d, on the basis of natural stilbene framework through medicinal chemistry approaches. This novel PELI1 inhibitor 3d showed potent binding affinity to PELI1 (Kd 8.2 μM) in fluorescence quenching assay, and markedly interrupted the interaction of PELI1 and SNAIL/SLUG confirmed by co-immunoprecipitation. Moreover, 3d exhibited potent antitumor activity in inhibiting tumor cell migration in scratch wound healing assay without affecting cell proliferation in vitro, and down-regulated the downstream EMT-effectors of PELI1 as assessed by western blotting. In the experimental lung metastasis model, 3d showed anti-TNBC metastasis efficacy without observable toxicity in vivo.

NUAK1 promotes tumor metastasis through upregulating slug transcription in esophageal squamous cell carcinoma

BackgroundMetastasis is still a major cause of poor pathological outcome and prognosis in esophageal squamous cell carcinoma (ESCC) patients. NUAK1 has been reported highly expressed in many human cancers and is associated with the poor prognosis of cancer patients. However, the role of NUAK1 and its underlying signaling mechanism in ESCC metastasis remain unclear.MethodsExpression of NUAK1 in ESCC was detected by real-time quantitative RT-PCR (qRT-PCR), Western blotting and immunohistochemical staining. MTT, colony formation, wound-healing and transwell assays were used to determine the role NUAK1 in vitro. Metastasis was evaluated by use of an experimental pulmonary metastasis model in BALB/c-nu/nu mice. The mechanisms were assessed by using coimmunoprecipitation, immunofluorescence and dual-luciferase reporter gene experiments.ResultsNUAK1 was highly expressed in ESCC tissues compared with the adjacent normal esophageal epithelial tissues. Moreover, the elevated expression of NUAK1 positively correlated with tumor invasion depth, lymph node metastasis, pathological TNM stage, and poor survival in ESCC patients. Further experiments showed that NUAK1 overexpression did not change the cell viability and colony formation of ESCC cells, while remarkably promoted the migration and invasion in vitro and experimental pulmonary metastasis in vivo. Mechanistically, NUAK1 enhanced the transcription level of Slug, which enhanced the migratory and invasive capability of ESCC cells. Consistently, silencing Slug almost completely diminished the migration and invasion of NUAK1-overexpressing ESCC cells. Further studies demonstrated that NUAK1 upregulated the transcription activity of Slug through activating the JNK/c-Jun pathway.ConclusionThese results demonstrated that NUAK1 promoted the metastasis of ESCC cells through activating JNK/c-Jun/Slug signaling, indicating NUAK1 is a promising therapeutic target for metastatic ESCC.

Experimental Peritoneal Metastasis Model: Which Type of Rodents Should we Choose, and which Method Should we Perform for the Intraperitoneal Inoculation of Tumor Cells?

Experimental Peritoneal Metastasis Model: Which Type of Rodents Should we Choose, and which Method Should we Perform for the Intraperitoneal Inoculation of Tumor Cells?

RETRACTED ARTICLE: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

Triple‑negative breast cancer (TNBC), a highly metastatic subtype of breast cancer, and it has the worst prognosis among all subtypes of breast cancer. However, no effective systematic therapy is currently available for TNBC metastasis. Therefore, novel therapies targeting the key molecular mechanisms involved in TNBC metastasis are required. The present study examined whether the expression levels of human epidermal growth factor receptor 3 (HER3) were associated with the metastatic phenotype of TNBC, and evaluated the potential of HER3 as a therapeutic target invitro and invivo. A new highly metastatic 4T1 TNBC cell line, termed 4T1‑L8, was established. The protein expression levels in 4T1‑L8 cells were measured using luminex magnetic bead assays and western blot analysis. The HER3 expression levels and distant metastasis‑free survival (DMFS) in TNBC were analyzed using Kaplan‑Meier Plotter. Transwell migration and invasion assays were performed to detect migration and invasion. The anti‑metastatic effects were determined in an experimental mouse model of metastasis. The results revealed that the increased expression of the HER3/Akt/mTOR pathway was associated with a greater level of cell migration, invasion and metastasis of TNBC cells. In addition, it was found that high expression levels of HER3 were associated with a poor DMFS. The inhibition of the HER3/Akt/mammalian target of rapamycin (mTOR) pathway decreased the migration, invasion and metastasis of TNBC cells by decreasing the expression of C‑X‑C chemokine receptor type 4 (CXCR4). Furthermore, treatment of metastatic TNBC cells with everolimus inhibited their migration, invasion and metastasis by decreasing CXCR4 expression. Thus, targeting the HER3/Akt/mTOR pathway opens up a new avenue for the development of therapeutics against TNBC metastasis; in addition, everolimus may prove to be an effective therapeutic agent for the suppression of TNBC metastasis.

Targeting Trop2 by Bruceine D suppresses breast cancer metastasis by blocking Trop2/β-catenin positive feedback loop

IntroductionTumor-associated calcium signal transducer 2 (Trop2) has been used as a transport gate for cytotoxic agents into cells in antibody–drug conjugate designs because of its expression in a wide range of solid tumors. However, the specific role of Trop2 itself in breast cancer progression remains unclear and small molecules targeting Trop2 have not yet been reported. ObjectivesTo screen small molecules targeting Trop2, and to reveal its pharmacological effects and the molecular mechanisms of action. MethodsSmall molecule targeting Trop2 was identified by cell membrane chromatography, and validated by cellular thermal shift assay and point mutation analyses. We investigated the pharmacological effects of Trop2 inhibitor using RNA-seq, human foreskin fibroblast (HFF)-derived extracellular matrix (ECM), Matrigel drop invasion assays, colony-forming assay, xenograft tumor model, 4T1 orthotopic metastasis model and 4T1 experimental metastasis model. The molecular mechanism was determined using immunoprecipitation, mass spectrometry, immunofluorescence, immunohistochemistry and Western blotting. ResultsHere we identified Bruceine D (BD) as the inhibitor of Trop2, and demonstrated anti-metastasis effects of BD in breast cancer. Notably, Lys307 and Glu310 residues of Trop2 acted as critical sites for BD binding. Mechanistically, BD suppressed Trop2-induced cancer metastasis by blocking the formation of Trop2/β-catenin positive loop, in which the Trop2/β-catenin complex prevented β-catenin from being degraded via the ubiquitin-proteosome pathway. Destabilized β-catenin caused by BD reduced nucleus translocation, leading to the reduction of transcription of Trop2, the reversal of epithelial-mesenchymal transition (EMT) process, and the inhibition of ECM remodeling, further inhibiting cancer metastasis. Additionally, the inhibitory effects of BD on lung metastatic colonization and the beneficial effects of BD on prolongation of survival were validated in 4T1 experimental metastasis model. ConclusionsThese results support the tumor-promoting role of Trop2 in breast cancer by stabilizing β-catenin in Trop2/β-catenin positive loop, and suggest Bruceine D as a promising candidate for Trop2 inhibition.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway

BackgroundLiver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PurposeThis study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. MethodsThe anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. ResultsThe invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. ConclusionThis study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Anti‑metastatic activity of aromatic aminomethylidenebisphosphonates in a mouse model of 4T1 cell‑derived breast cancer.

Breast cancer is one of the major causes of cancer‑related mortality among women worldwide. It metastasizes to distant organs, particularly to bone tissue. Nitrogen‑containing bisphosphonates are mainly used as an adjuvant therapy to inhibit skeletal‑related events; however, there is increasing evidence to suggest that these compounds also exert antitumor effects. In previous studies, the authors synthesized two novel aminomethylidenebisphosphonates (BPs), namely benzene‑1,4‑bis[aminomethylidene(bisphosphonic)] acid (WG12399C) and naphthalene‑1,5‑bis[aminomethylidene(bisphosphonic)] acid (WG12592A). Both BPs exhibited notable antiresorptive activity in a mouse model of osteoporosis. The present study aimed to assess the invivo anticancer activity of WG12399C and WG12592A in 4T1 breast adenocarcinoma model. WG12399C exerted an anti‑metastatic effect by reducing the number of spontaneous lung metastases by ~66% in comparison to the control. In the experimental metastasis model of 4T1‑luc2‑tdTomato cells, this compound reduced the incidence of tumor metastases in the lungs by approximately half in comparison to the control. Both WG12399C and WG12595A also significantly reduced the size and/or number of bone metastatic foci. Their pro‑apoptotic and anti‑proliferative activity may, at least in part, explain the observed effects. Incubation with WG12399C induced an almost 6‑fold increase in caspase‑3 activity in 4T1 cells. Moreover, cells treated with WG12399C or WG12595A exhibited a 2‑fold reduction in invasiveness through Matrigel. Furthermore, both the BPs were able to sensitize the 4T1 cells to cytostatics. In summary, the results of the present study indicate that the examined aminomethylidene‑BPs may be of particular interest in the context of combined treatment in breast cancer therapy.

Investigation of the effectiveness of hyperthermic intraperitoneal chemotherapy in experimental colorectal peritoneal metastasis model.

In our study, we aimed to (1) create a peritoneal metastasis (PM) model in nude mice, administer intraperitoneal chemotherapy using the peritoneal infusion pump we developed in this model, and (2) compare the efficacy of intraperitoneal chemotherapy using various drugs at different temperatures. The peritoneal metastasis model was established in nude mice using the CC531 colon carcinoma cell line. Models with peritoneal metastasis (PM) were randomized into four groups of seven animals each: Group 1, control group (n=7); Group 2, normothermic intraperitoneal chemotherapy (NIPEC) with mitomycin C(MMC) (n=7); Group 3, hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (n=7), and Group 4, NIPEC with 5-fluorouracil (5-FU). Tumor development was achieved in all animals. While the tumor burden decreased significantly in the treatment Group 3 (p=0.034), no significant difference was found in the other groups. In the PM mouse model, hyperthermic intraperitoneal administration of MMC had the highest tumoricidal effect. Our PM model provided a good opportunity to examine the efficacy of HIPEC and intraperitoneal infusion pump (IPIP). In future studies, we plan to evaluate efficacies of different drugs in the PM models we have created.

Multivalent dipeptidyl peptidase IV fragment-nanogold complex inhibits cancer metastasis by blocking pericellular fibronectin

Inhibition of cancer metastasis is a fundamental challenge in cancer treatment. We have previously shown that metastasis of cancer cells in the lung is critically promoted by the interaction between the superficial dipeptidyl peptidase IV (DPP IV) expressed on lung endothelial cells and the pericellular polymeric fibronectin (polyFN) of circulating cancer cells. In the present study, we aimed to search for DPP IV fragments with high avidity to polyFN and develop FN-targeted gold nanoparticles (AuNPs) conjugated with DPP IV fragments for treating cancer metastasis. We first identified a DPP IV fragment encompassing amino acids 29-130 of DPP IV, designated DP4A, which contained FN-binding sites and could specifically bind to FN immobilized on gelatin agarose beads. Furthermore, we conjugated maltose binding protein (MBP)-fused DP4A proteins to AuNPs for fabricating a DP4A-AuNP complex and evaluated its FN-targeted activity in vitro and anti-metastatic efficacy in vivo. Our results show that DP4A-AuNP exhibited higher binding avidity to polyFN than DP4A by 9 folds. Furthermore, DP4A-AuNP was more potent than DP4A in inhibiting DPP IV binding to polyFN. In terms of polyFN-targeted effect, DP4A-AuNP interacted with FN-overexpressing cancer cells and was endocytosed into cells 10 to 100 times more efficiently than untargeted MBP-AuNP or PEG-AuNP with no noticeable cytotoxicity. Furthermore, DP4A-AuNP was superior to DP4A in competitive inhibition of cancer cell adhesion to DPP IV. Confocal microscopy analysis revealed that binding of DP4A-AuNP to pericellular FN induced FN clustering without altering its surface expression on cancer cells. Notably, intravenous treatment with DP4A-AuNP significantly reduced metastatic lung tumor nodules and prolonged the survival in the experimental metastatic 4T1 tumor model. Collectively, our findings suggest that the DP4A-AuNP complex with potent FN-targeted effects may have therapeutic potential for prevention and treatment of tumor metastasis to the lung.

Vascular endothelial-derived Von Willebrand factor inhibits lung cancer progression through the αvβ3/ERK1/2 axis

Lung cancer remains a common malignant tumor causing death due to the rapid industrialization and serious pollution of the environment. The Von Willebrand Factor (vWF) protein is an endothelial marker and is widely used to diagnose cancer and other inflammations, however its exact mechanism of action remains largely unexplored. In particular, how it plays two opposing roles in tumor development is not clear. Our study aimed to the impact of endothelial-derived vWF on tumor development by co-culturing human umbilical vein endothelial cells (HUVECs) with lung cancer cells (95D and A549). A knockdown of endothelial-derived vWF assisted lung cancer cell in proliferation, migration and inhibited apoptosis in vitro, while overexpression of endothelial-derived vWF inhibited the proliferation, migration and induced apoptosis of lung cancer cells. The results of further experiments indicated that the vWF secreted by endothelial cells could affect lung cancer cell migration and apoptosis via its binding to integrin αvβ3 on the surface of lung cancer cells. Furthermore, a novel finding was the fact that endothelial-derived vWF inhibited lung cancer cell apoptosis by phosphorylating ERK1/2. At the same time, we established experimental lung metastasis model and xenograft model in normal mice and vWF−/− mice, and found that knockout of vWF in mice significantly promoted lung cancer growth and metastasis. In conclusion, our research found that endothelial-derived vWF could directly combine to αvβ3 on the exterior of A549 and 95D, thereby mediating lung cancer proliferation, migration and apoptosis and inhibiting the development of lung cancer.

Dormancy in Breast Cancer.

The pattern of delayed recurrence in a subset of breast cancer patients has long been explained by a model that incorporates a variable period of cellular or tumor mass dormancy prior to disease relapse. In this review, we critically evaluate existing data to develop a framework for inferring the existence of dormancy in clinical contexts of breast cancer. We integrate these clinical data with rapidly evolving mechanistic insights into breast cancer dormancy derived from a broad array of genetically engineered mouse models as well as experimental models of metastasis. Finally, we propose actionable interventions and discuss ongoing clinical trials that translate the wealth of knowledge gained in the laboratory to the long-term clinical management of patients at a high risk of developing recurrence.

Abstract 2451: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Abstract Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis. Citation Format: Changhwan Yoon, Jun Lu, Yukyung Jun, Bang- Jin Kim, Jacob Till, Jong Hyun Kim, Sara Keshavjee, Sandra Ryeom, Sam Yoon. KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and 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 2451.