Orthotopic Transplantation Mouse Model Research Articles

Tumor microenvironment dynamics in oral cancer: unveiling the role of inflammatory cytokines in a syngeneic mouse model.

The process of cervical lymph node metastasis is dependent on the phenotype of the tumor cells and their interaction with the host microenvironment and immune system; conventional research methods that focus exclusively on tumor cells are limited in their ability to elucidate the metastatic mechanism. In cancer tissues, a specialized environment called the tumor microenvironment (TME) is established around tumor cells, and inflammation in the TME has been reported to be closely associated with the development and progression of many types of cancer and with the response to anticancer therapy. In this study, to elucidate the mechanism of metastasis establishment, including the TME, in the cervical lymph node metastasis of oral cancer, we established a mouse-derived oral squamous cell carcinoma cervical lymph node highly metastatic cell line and generated a syngeneic orthotopic transplantation mouse model. In the established highly metastatic cells, epithelial-mesenchymal transition (EMT) induction was enhanced compared to that in parental cells. In the syngeneic mouse model, lymph node metastasis was observed more frequently in tumors of highly metastatic cells than in parental cells, and Cyclooxygenase-2 (COX-2) expression and lymphatic vessels in primary tumor tissues were increased, suggesting that this model is highly useful. Moreover, in the established highly metastatic cells, EMT induction was enhanced compared to that in the parent cell line, and CCL5 and IL-6 secreted during inflammation further enhanced EMT induction in cancer cells. This suggests the possibility of a synergistic effect between EMT induction and inflammation. This model, which allows for the use of two types of cells with different metastatic and tumor growth potentials, is very useful for oral cancer research involving the interaction between cancer cells and the TME in tumor tissues and for further searching for new therapeutic agents.

Discovery of Novel Dual Inhibitors Targeting Mutant IDH1 and NAMPT for the Treatment of Glioma with IDH1Mutation.

The targeting of cancer cell intrinsic metabolism has emerged as a promising strategy for antitumor intervention. In the study, we identified the first-in-class small molecules that effectively inhibit both mutant isocitrate dehydrogenase 1 (mIDH1) and nicotinamide phosphoribosyltransferase (NAMPT), two crucial targets in cancer metabolism, through structure-based drug design. Notably, compound 23h exhibits excellent and balanced inhibitory activities against both mIDH1 (IC50 = 14.93 nM) and NAMPT (IC50 = 12.56 nM), leading to significant suppression of IDH1-mutated glioma cell (U87 MG-IDH1R132H) proliferation. Significantly, compound 23h has the ability to cross the blood-brain barrier (B/P ratio, 0.76) and demonstrates remarkable in vivo antitumor efficacy (20 mg/kg) in the U87 MG-IDH1R132H orthotopic transplantation mouse models without any notable toxicity. This proof-of-concept investigation substantiates the viability of discovering small molecules that concurrently target mIDH1 and NAMPT, providing valuable leads for the treatment of glioma and an efficient approach for the discovery of multitarget antitumor drugs.

Abstract 2190: Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach

Abstract Obesity is a highly prevalent metabolic disease and an established risk factor for colon cancer. However, critical biological pathways underpinning this relationship remain undetermined. Herein, we integrate human and murine colon tumor transcriptomic data to identify obesity-responsive pathways conserved between species. Human colon cancer samples (n=155) were collected as part of the clinical ColoCare Study, a prospective cohort study of patients newly diagnosed with colorectal cancer. Enrolled patients were diagnosed with stage I-III colon cancer and treatment naïve at time of tissue collection. Patients were classified as having a BMI <30 kg/m2 (i.e., non-obese) or BMI ≥30 kg/m2 (i.e., obese) at baseline. Age, sex, tumor stage, and collection site were adjusted for in all analyses. Patients were 63.3 ± 13.7 years, 51% male, and 34% had a BMI ≥30. C57BL/6J adult female mice were fed a low-fat control diet (Research Diets D12450J, n= 8) or a high-fat diet-induced obesity regimen (D12492, n=7) for ~23 weeks, at which time 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were orthotopically transplanted into the colonic submucosa via colonoscopy-guided injection. Obese mice weighed more than controls (controls 26.4 ± 3.7 g vs. DIO 45.9 ± 9.7 g) and had more mesenteric fat surrounding the colon (controls 201 ± 143 mg vs. 1176 ± 589 mg). Tumors were excised after 4 weeks of growth. RNA was extracted from bulk human tumor samples and from the epithelial-enriched EpCAM+ cell population in murine tumor samples for RNA sequencing. Genes differentially expressed between the reference and obese groups were identified using DESeq2. For gene set enrichment analysis, genes were preranked by log2 fold change for both human and mouse datasets. There were 76 and 132 differentially expressed genes (DEGs; FDRq<0.10) identified in the human and mouse datasets, respectively. None of these 208 DEGs were common to both datasets. Thirty-five Hallmark gene sets in the human dataset and 25 Hallmark gene sets in the mouse dataset were significantly enriched in the obese group; 18 of these gene sets were common to both mouse and human datasets. These overlapping pathways were largely related to inflammation (e.g., inflammatory response, IL6 JAK STAT3 signaling, TNFɑ signaling) and metabolism (e.g., cholesterol homeostasis, glycolysis, and fatty acid metabolism). Additional analysis revealed 204 genes common between the leading edges of both transcriptomic datasets. These transdisciplinary data suggest that obesity induces a set of metabolic and inflammatory processes and/or pathways in colon tumors which is conserved between mice and humans. Future studies will mechanistically evaluate the role of candidate genes and pathways in obesity-associated colorectal carcinogenesis in our AKPS orthotopic organoid transplant mouse model. Citation Format: Elaine M. Glenny, Tengda Lin, Victoria M. Bandera, Biljana Gigic, Aik Choon Tan, Babak Mirminachi, Saratchandra S. Khumukcham, Alessandro Carpanese, Christy A. Warby, Olena Aksonova, Caroline Himbert, Jennifer Ose, Chris Stubben, David Nix, Kenneth Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Alexander Brobeil, Martin A. Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Jatin Roper, Cornelia M. Ulrich, Stephen D. Hursting. Transcriptomic signatures of obesity-driven colon cancer: A multi-species transdisciplinary approach [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 2190.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma [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 2367.

YY1 regulates the proliferation and invasion of triple-negative breast cancer via activating PLAUR.

It is well-established that breast cancer is a highly prevalent malignancy among women, emphasizing the need to investigate mechanisms underlying its pathogenesis and metastasis. In this study, the Gene Expression Omnibus (GEO) database was utilized to conduct differential expression analysis in breast cancer and adjacent tissues. Upregulated genes were selected for prognostic analysis of breast cancer. The expression of urokinase plasminogen activator receptor (uPAR), also known as PLAUR, was assessed using RT-qPCR and western blot. Immunofluorescence staining was employed to determine PLAUR localization. Various cellular processes were analyzed, including proliferation, migration, invasion, apoptosis, and cell cycle. Bioinformatics analysis was used to predict transcription factors of PLAUR, which were subsequently validated in a double luciferase reporter gene experiment. Rescue experiments confirmed the impact of PLAUR on the proliferation, apoptosis, and migration of MDA-MB-231 cells. Furthermore, the effects of PLAUR were evaluated in an orthotopic tumor transplantation and lung metastasis nude mouse model. Our findings substantiated the critical involvement of PLAUR in the progression of triple-negative breast cancer (TNBC) in vitro and among TNBC patients with a poor prognosis. Additionally, we demonstrated Yin Yang-1 (YY1) as a notable transcriptional regulator of PLAUR, whose activation could transcriptionally enhance the proliferation and invasion capabilities of TNBC cells. We also identified the downstream mechanism of PLAUR associated with PLAU, focal adhesion kinase (FAK), and AKT. Overall, these findings offer a novel perspective on PLAUR as a potential therapeutic target for TNBC.

Semaphorin 3 C enhances putative cancer stemness and accelerates peritoneal dissemination in pancreatic cancer

PurposeSemaphorins, axon guidance cues in neuronal network formation, have been implicated in cancer progression. We previously identified semaphorin 3 C (SEMA3C) as a secreted protein overexpressed in pancreatic ductal adenocarcinoma (PDAC). We, therefore, hypothesized that SEMA3C supports PDAC progression. In this study, we aimed to investigate the clinical features of SEMA3C, especially its association with chemo-resistance and peritoneal dissemination.MethodsIn resected PDAC tissues, we assessed the relationship between SEMA3C expression and clinicopathological features by immunohistochemistry. In vitro studies, we have shown invasion assay, pancreatosphere formation assay, colony formation assay, cytotoxicity assay, and activation of SEMA3C downstream targets (c-Met, Akt, mTOR). In vivo, we performed a preclinical trial to confirm the efficacy of SEMA3C shRNA knockdown and Gemcitabine and nab-Paclitaxel (GnP) in an orthotopic transplantation mouse model and in peritoneal dissemination mouse model.ResultsIn resected PDAC tissues, SEMA3C expression correlated with invasion and peritoneal dissemination after surgery. SEMA3C promoted cell invasion, self-renewal, and colony formation in vitro. We further demonstrated that SEMA3C knockdown increased Gem-induced cytotoxicity by suppressing the activation of the Akt/mTOR pathway via the c-Met receptor. Combination therapy with SEMA3C knockdown and GnP reduced tumor growth and peritoneal dissemination.ConclusionsSEMA3C enhances peritoneal dissemination by regulating putative cancer stemness and Gem resistance and activates phosphorylation of the Akt/mTOR pathway via c-Met. Our findings provide a new avenue for therapeutic strategies in regulating peritoneal dissemination during PDAC progression.

Antiproliferative and apoptotic effects of telmisartan in human glioma cells

Glioblastoma is the most common primary central nervous system tumor in adults. Angiotensin II receptor blockers (ARBs) are broadly applied to treat hypertension. Moreover, research has revealed that ARBs have the capacity to suppress the growth of several cancer types. In this study, we assessed the effects of three ARBs with the ability to cross the blood brain barrier (telmisartan, valsartan and fimasartan) on cell proliferation in three glioblastoma multiforme (GBM) cell lines. Telmisartan markedly suppressed the proliferation, migration, and invasion of these three GBM cell lines. Microarray data analysis revealed that telmisartan regulates DNA replication, mismatch repair, and the cell cycle pathway in GBM cells. Furthermore, telmisartan induced G0/G1 phase arrest and apoptosis. The bioinformatic analysis and western blotting results provide evidence that SOX9 is a downstream target of telmisartan. Telmisartan also suppressed tumor growth in vivo in an orthotopic transplant mouse model. Therefore, telmisartan is a potential treatment for human GBM.

ERβ promoted invadopodia formation-mediated non-small cell lung cancer metastasis via the ICAM1/p-Src/p-Cortactin signaling pathway.

In a previous study, our research group observed that estrogen promotes the metastasis of non-small cell lung cancer (NSCLC) through the estrogen receptor β (ERβ). Invadopodia are key structures involved in tumor metastasis. However, it is unclear whether ERβ is involved in the promotion of NSCLC metastasis through invadopodia. In our study, we used scanning electron microscopy to observe the formation of invadopodia following the overexpression of ERβ and treatment with E2. In vitro experiments using multiple NSCLC cell lines demonstrated that ERβ can increase the formation of invadopodia and cell invasion. Mechanistic studies revealed that ERβ can upregulate the expression of ICAM1 by directly binding to estrogen-responsive elements (EREs) located on the ICAM1 promoter, which in turn can enhance the phosphorylation of Src/cortactin. We also confirmed these findings in vivo using an orthotopic lung transplantation mouse model, which validated the results obtained from the in vitro experiments. Finally, we examined the expressions of ERβ and ICAM1 using immunohistochemistry in both NSCLC tissue and paired metastatic lymph nodes. The results confirmed that ERβ promotes the formation of invadopodia in NSCLC cells through the ICAM1/p-Src/p-Cortactin signaling pathway.

Anti-tumor necrosis factor-alpha monoclonal antibody suppresses colorectal cancer growth in an orthotopic transplant mouse model.

The risk of malignant tumor progression has been a concern associated with the use of anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNFα mAb). On the contrary, recent observational studies have reported negatively on this risk and instead suggested that anti-TNFα mAb acts as a tumor suppressor in inflammatory carcinogenesis models and subcutaneous transplant models of colorectal cancer. However, no consensus has been established regarding the actual effects of anti-TNFα mAb on malignant tumors. Here, we aimed to evaluate, for the first time, the effect of anti-TNFα mAb on the tumor microenvironment in the absence of intestinal inflammation in a colorectal cancer orthotopic transplant mouse model suitable for tumor microenvironment assessment. The orthotopic transplantation model was developed by transplanting CT26 cells into the cecum of BALB/c mice. Changes in tumor size and weight were recorded 3 weeks after transplantation, and the tumor microenvironment was assessed via RNA sequencing and immunohistological staining. In the orthotopic transplant model, the administration of anti-TNFα mAb led to a reduction in colorectal cancer. The RNA sequencing analysis showed upregulation of immune-related pathways and apoptosis and suppression of stromal- and tumor growth-related pathways. Additionally, Gene Ontology analysis showed inhibition of angiogenesis. Immunohistochemical staining showed inhibition of tumor growth, increase in apoptosis, suppression of stromal response, suppression of angiogenesis, enhancement of tumor immunity, and reduction in the number of tumor-associated macrophages. Anti-TNFα mAb acts as an inhibitor of tumor progression in the tumor microenvironment of a colorectal cancer orthotopic transplant mouse model.

Living on the edge: how stromal cells restrict liver cancer invasion

The global incidence of primary liver cancer is increasing and currently ranks amongst the top-five causes of premature death from cancer annually. Like in many solid tumours, liver cancer cells are closely associated with stromal cells, known as non-parenchymal cells (NPCs), in the liver, including fibroblasts and endothelial cells, which normally support liver function and structure. Although single-cell RNA sequencing has begun to unpick the interactions between tumour cells and this non-epithelial microenvironment, these -omic approaches cannot mechanistically resolve how the non-parenchymal stroma interacts with tumour cells, nor can they resolve whether a fibroblast exerts differential outcomes on mutant versus healthy epithelial cells.In this issue of Disease Models & Mechanisms, Liqin Zhu and colleagues describe a novel in vitro microwell culture system to co-culture NPCs with either healthy hepatocytes or tumour cells of intrahepatic cholangiocarcinoma (iCCA), an aggressive type of primary liver cancer. When the authors co-cultured NPCs with primary hepatocytes, the NPCs supported hepatocyte spheroid growth, maintaining the phenotype of the healthy hepatocytes in culture. Interestingly, when cultured with mutant iCCA cells, NPCs supressed the growth of cancer spheroids, suggesting a divergent role of NPCs in tumour growth.To understand the role of these stromal cells in tumour growth further, the authors then co-cultured healthy hepatocytes and tumour cells in their microwell system and found that tumour cells invade and kill healthy hepatocytes. The addition of NPCs to this system significantly limited this process, and in vitro cancer cells were constrained, no longer killing healthy hepatocytes. Following on from this, the authors used an orthotopic transplantation mouse model of liver cancer and liver tumour samples from patients to show that activated myofibroblasts localised at the interface between tumour and normal tissue. Together, the data suggest that, in liver cancer, NPCs, in particular myofibroblasts, form a physical barrier that limits the ability of cancer cells to invade and damage normal tissue.Targeting the tumour microenvironment to limit disease progression is an attractive therapeutic prospect that could be agnostic to the underlying genetic and molecular heterogeneity seen in cancer. However, as NPCs have previously been shown to provide pro-proliferative signals in cancer and modify the intratumoral immune landscape, more research is required to understand the nuanced NPC-epithelial cell relationship in cancer. We should carefully consider the diverse and potentially conflicting roles of NPCs in tumour progression, and advanced in vitro systems, such as those developed by Zhu and colleagues, will help us decipher this.

Sufentanil combined with parecoxib sodium inhibits proliferation and metastasis of HER2-positive breast cancer cells and regulates epithelial-mesenchymal transition.

Sufentanil combined with parecoxib sodium is a commonly used postoperative medication for cancer patients. However, the effects of this combination therapy on human epidermal growth factor receptor-2 (HER2)-positive breast cancer cells have still remained elusive. This study aimed to investigate the effects and potential mechanisms of sufentanil combined with parecoxib sodium on HER2-positive breast cancer cells. The cell counting kit-8 (CCK-8), colony formation, flow cytometry, scratch, transwell invasion, and angiogenesis assays were used to assess the proliferation, cell cycling, migration, invasion, and angiogenesis of HER2-positive breast cancer BT474 cells. Western blot assay was employed for detecting the expression levels of proteins involved in the cell cycle, migration, invasion, angiogenesis, and epithelial-mesenchymal transition (EMT). The in vivo effects of tumor growth and metastasis were examined by establishing an orthotopic transplantation mouse model of HER2-positive breast cancer (MMTV-PyMT). Functional assays indicated that sufentanil combined with parecoxib sodium induced blockade of HER2-positive breast cancer BT474 cells in the G1 phase of the cell cycle and inhibited cell proliferation, migration, angiogenesis, and invasion in vitro. Western blot assay revealed that sufentanil combined with parecoxib sodium downregulated the expression levels of cyclin D1, matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), vascular endothelial growth factor A (VEGFA), and EMT-related proteins (N-cadherin, Vimentin, and Snail), while up-regulated the expression level of E-cadherin in BT474 cells. In addition, it was found that sufentanil combined with parecoxib sodium inhibited tumor growth and metastasis in the orthotopic transplantation mouse model of HER2-positive breast cancer. Sufentanil combined with parecoxib sodium inhibited HER2-positive breast cancer progression, including cell proliferation, cell cycle, migration, invasion, and angiogenesis, and regulated EMT.

HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

BackgroundHepatic inflammation is a common initiator of liver diseases and considered as the primary driver of hepatocellular carcinoma (HCC). However, the precise mechanism of inflammation-induced HCC development and immune evasion remains elusive and requires extensive investigation. This study sought to identify the new target that is involved in inflammation-related liver tumorigenesis.MethodsRNA-sequencing (RNA-seq) analysis was performed to identify the differential gene expression signature in primary human hepatocytes treated with or without inflammatory stimulus. A giant E3 ubiquitin protein ligase, HECT domain and RCC1-like domain 2 (HERC2), was identified in the analysis. Prognostic performance in the TCGA validation dataset was illustrated by Kaplan–Meier plot. The functional role of HERC2 in HCC progression was determined by knocking out and over-expressing HERC2 in various HCC cells. The precise molecular mechanism and signaling pathway networks associated with HERC2 in HCC stemness and immune evasion were determined by quantitative real-time PCR, immunofluorescence, western blot, and transcriptomic profiling analyses. To investigate the role of HERC2 in the etiology of HCC in vivo, we applied the chemical carcinogen diethylnitrosamine (DEN) to hepatocyte-specific HERC2-knockout mice. Additionally, the orthotopic transplantation mouse model of HCC was established to determine the effect of HERC2 during HCC development.ResultsWe found that increased HERC2 expression was correlated with poor prognosis in HCC patients. HERC2 enhanced the stemness and PD-L1-mediated immune evasion of HCC cells, which is associated with the activation of signal transducer and activator of transcription 3 (STAT3) pathway during the inflammation-cancer transition. Mechanically, HERC2 coupled with the endoplasmic reticulum (ER)-resident protein tyrosine phosphatase 1B (PTP1B) and limited PTP1B translocation from ER to ER-plasma membrane junction, which ameliorated the inhibitory role of PTP1B in Janus kinase 2 (JAK2) phosphorylation. Furthermore, HERC2 knockout in hepatocytes limited hepatic PD-L1 expression and ameliorated HCC progression in DEN-induced mouse liver carcinogenesis. In contrast, HERC2 overexpression promoted tumor development and progression in the orthotopic transplantation HCC model.ConclusionOur data identified HERC2 functions as a previously unknown modulator of the JAK2/STAT3 pathway, thereby promoting inflammation-induced stemness and immune evasion in HCC.

CCNE1 is a potential target of Metformin for tumor suppression of ovarian high-grade serous carcinoma

ABSTRACT High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer, accounting for 70%–80% of mortality. However, the treatment of HGSOC has improved little in the past few decades. Metformin is the first-line medication for the treatment of type 2 diabetes and has now gained more attention in cancer treatment. In this study, we sought to identify potential hub genes that metformin could target in the treatment of HGSOC. We downloaded GSE69428 and GSE69429 in the Gene Expression Omnibus database and performed the bioinformatics analysis. Subsequently, we analyzed the effect of Metformin in HGSOC through biological experiments. Molecular simulation docking was used to predict the interaction of Metformin and CCNE1. We chose CCNE1 for the study based on bioinformatics analysis, literature studies, and preliminary data. We evaluated that CCNE1 is overexpressed in HGSOC tissues and found that HGSOC cells with high CCNE1 expression increase sensitivity to Metformin treatment in the analysis of cell proliferation and anchorage-independent growth. Metformin could inhibit the expression of CCNE1, which is associated with the anti-proliferative effect of tumor cells. Moreover, Metformin could ameliorate the tumor growth in syngeneic orthotopic transplantation mouse models and xenograft tumorigenesis models. Furthermore, molecular simulation docking showed that Metformin may bind to CCNE1 protein, suggesting that CCNE1 could be a potential target for Metformin. Our data revealed that Metformin has antitumor effects on ovarian cancer and CCNE1 could be a potential target for Metformin.

Detecting and monitoring tumors in orthotopic colorectal liver metastatic animal models with high-resolution ultrasound.

The ability to noninvasively detect and monitor the growth of orthotopic liver transplantation tumors is critical for replicating advanced colorectal cancer liver metastases (CRLMs) in animal models. We assessed the use of high-resolution ultrasound (HRU) to monitor CRLMs transplanted using various cell concentrations. Sixty BALB/c female mice were randomly divided into 3 groups, and murine colonic CT26 cells were injected into the left liver lobe at concentrations of 1 × 102 (group 1), 1 × 103 (group 2), or 1 × 104 (group 3). Tumor presentation, location, number, size, shape, and echogenicity were assessed daily with 24-MHz center frequency HRU starting 6days after injection. Animals were sacrificed when the largest tumor was ≥ 1cm in diameter. Sensitivity, specificity, and area under curve (AUC) of CRLMs diagnosed with HRU were calculated using receiver operating characteristic curve analysis. In group 1, 94% of mice formed < 5 tumors, and 41% formed a single tumor. Tumors were first detected with HRU on day 12 in group 1, day 10 in group 2, and day 7 in group 3; tumor volume doubling times were 14-15days, 11-12days, and 7-8days, respectively. With a long diameter threshold of 2.4mm, diagnostic sensitivity and specificity of HRU were 94.1% and 88.7%, respectively, and the AUC was 0.962. These findings suggest that HRU can be used to accurately detect and monitor the growth of CRLMs in an orthotopic transplantation mouse model, especially when a lower concentration of cells is used.

Obesity Strongly Promotes Growth of Mouse MC38 Colon Cancer in an Orthotopic-syngeneic C57BL/6 Mouse Model

Obesity is a major risk factor for colorectal cancer. The MC38 mouse colon-cancer cell line is a versatile syngeneic model of colon cancer in C57BL/6 mice. In the present study, the influence of a high-fat diet (HFD) on the growth of the MC38 mouse colon-cancer cell line was examined in an orthotopic-transplantation syngeneic model in C57BL/6 mice. Five 6-week-old C57BL/6 male mice were fed a control diet (CD, 6.5% fat) or HFD (34.3% fat) for eight weeks. Then, a 2 mm3 fragment of a subcutaneous MC38 tumor was attached to the surface of the cecum of C57BL/6 mice with a single stitch using a 7-0 suture to establish an orthotopic-transplantation model. Each group continued their initial diet for 17 days. The HFD group had more than twice the tumor volume and tumor weight than the CD group (p=0.021 and p=0.014, respectively). HFD-induced obesity strongly increased MC38 colon-cancer progression in a C57BL/6 orthotopic-transplantation mouse model. The present study emphasizes the detrimental effect of obesity on coloncancer progression.

Reduction-Responsive and Multidrug Deliverable Albumin Nanoparticles: An Antitumor Drug to Abraxane against Human Pancreatic Tumor-Bearing Mice.

Many macromolecular antitumor drugs were developed based on the enhanced permeability and retention (EPR) effect, for example, albumin-bound paclitaxel nanoparticles (nab-PTX and Abraxane) and pegylated liposomal doxorubicin (Doxil). However, these EPR effect-based therapeutic systems are less effective in malignant tumors with low vascular permeability, such as pancreatic tumors. Because the EPR effect depends on nanoparticles' size, we first determined nanoparticles' size associated with a high tumor-targeting rate in a human pancreatic tumor xenograft model with low vascular permeability. Abraxane appears to behave as an albumin monomer (7 nm) in the blood circulation following intravenous injection. The in vitro and in vivo tumor-targeted delivery and antitumor activity of PTX-loaded albumin nanoparticles were significantly improved by optimizing the mean nanoparticle diameter to 30 nm. Furthermore, nitric oxide was added to 30 nm PTX-loaded albumin nanoparticles to examine the feasibility of albumin nanoparticles as a platform for multiple drug delivery. Their antitumor effect was evaluated in an orthotopic transplantation mouse model of a human pancreatic tumor. The nitric oxide PTX-loaded 30 nm albumin nanoparticle treatment on model mice achieved a significantly higher survival rate than Abraxane treatment. These findings suggest that 30 nm albumin nanoparticles have a high therapeutic effect as a useful platform for multiple drugs against human pancreatic tumors.

Gut Fecal Microbiota Transplant in a Mouse Model of Orthotopic Rectal Cancer.

The gut microbiota is reported to play an important role in carcinogenesis and the treatment of CRC. SW480 and SW620 colon cancer cells integrated with infrared fluorescent proteins were injected into the rectal submucosa of nude mice. In the subsequent 30 days, we observed tumor growth weekly using an in vivo imaging system. The bacterial solution was infused anally into the mice to perform bacterial transplant. Phosphate-buffered saline, Acinetobacter lwoffii, and Bifidobacterium longum solutions were infused individually. The 16S ribosomal DNA (rDNA) and polymerase chain reaction of murine feces were investigated to confirm the colonization of target bacteria. In the SW620 orthotopic xenograft rectal cancer model, 4 of 5 mice developed rectal cancer by 30 days after submucosal injection. In the SW480 orthotopic xenograft rectal cancer model, 2 of 6 mice developed rectal cancer by 30 days after submucosal injection. For the 16S rDNA analysis, the mice receiving the bacterial solution infusion demonstrated positive findings for A. lwoffii and B. longum. With the successful establishment of a mouse model of orthotopic rectal cancer and transplant of target bacteria, we can further explore the relationship between gut microbiota and CRC. The role of fecal microbiota transplant in the treatment and alleviation of adverse events of chemotherapy in CRC could be clarified in subsequent studies.

Abstract 4179: E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Natural products have been a rich source of inspiration for drug discovery as demonstrated by the fact that over one-third of current therapeutic agents are natural products or compounds derived from them. Particularly, when the supply of a natural product from the natural source is limited, organic synthesis can offer a solution. However, with increasing structural complexity, this approach becomes exponentially more challenging, as a synthesis must meet various requirements, including high overall efficiency, scalability, cost-effectiveness, and product purity as well as conforming to good manufacturing practice (GMP) regulations. Despite the outstanding in vivo antitumor activity in mice, the limited supply from the natural sources has prevented drug development based on intact halichondrins. Methods: We successfully synthesized 19.5 g of C52-halichondrin-B alcohol with 99.84% purity via a total synthesis. From 15.0 g of this material, we obtained 11.5 g of C52-halichondrin-B amine (E7130) with 99.81% purity under GMP conditions. With totally synthetic E7130, a novel microtubule dynamics inhibitor, we studied the activities in both of in vitro and in vivo. The inhibitory activity of E7130 towards tubulin polymerization was analyzed in the cell-free system in which tubulin polymerization could be monitored by fluorescence enhancement due to the incorporation of a fluorescence reporter into the microtubules during polymerization. We analyzed in vivo antitumor activities using both a human cancer cell line orthotopic transplantation mouse model and subcutaneous xenograft models. The immunohistochemical analyses were performed with tumor tissues collected from mouse models to analyze histological changes after treatment with E7130. Results: E7130 is a novel microtubule dynamics inhibitor with exceedingly potent in vitro and in vivo anticancer activities. Significantly, E7130 not only is cytotoxic, but can also increase CD31-positive endothelial cells in tumors and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. Notably, this dual activity has been recognized for the first time through this study. According to these unique tumor microenvironment ameliorative effects, E7130 can augment the effect of other antitumor treatments in mouse models. In particular, a dose of 90 µg/kg, one-half of the maximum tolerated dose in mice, showed a prominent combinational effect with cetuximab, which suggests that E7130 has a different mechanism than other microtubule-targeted drugs. Conclusions: E7130 ameliorates the tumor microenvironment to improve cancer treatment when used in combination with other compounds. The data provide compelling evidence that E7130 is a promising molecular-targeting anticancer agent. Citation Format: Satoshi Kawano, Takanori Abe, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4179.

PATH-36. MACHINE LEARNING TO DETECT GLIOMAS IN URINE-BASED LIQUID BIOPSY

Abstract BACKGROUND Diffuse gliomas are the most common primary malignant brain tumors, whose overall prognosis is quite dismal. Tumor-cell-secreted extracellular vesicles (EVs) participate in physiological and pathological processes and have potential applications to diagnostics of malignant tumors including diffuse gliomas. Because urine is less invasive to collect, development of early diagnosis based on urine EVs is eagerly awaited. In this study, we captured urine EVs of patients with gliomas efficiently with the nanowire device and compared expression profile of microRNAs (miRNAs) within urine EVs with that of healthy donors to identify diagnostic accuracy by a machine learning algorithm. METHODS 62 patients with diffuse gliomas, including 27 glioblastoma and 35 lower grade gliomas, and 100 healthy donors were analyzed, along with orthotopic transplant mouse model. Urinary EVs were obtained with the nanowire device which could collect EVs more efficiently than the conventional ultracentrifugation method (Yasui et.al., Science Adv.2017). Machine learning methods were performed to select the miRNAs which could distinguish patients with gliomas from healthy control. RESULTS More than 2400 miRNAs were obtained from all urine samples. We identified miRNA panels that provided high diagnostic accuracy of diffuse gliomas (92.5%). There were 440 miRNAs whose expression increased by more than 1.5 fold (p&lt; 0.05) as compared to healthy donor samples (glioma-upregulated miRNAs), whereas the expression of 87 miRNAs decreased to less than 2/3-fold (p&lt; 0.05) (glioma-downregulated miRNAs). Mouse miRNAs which were homologous to glioma-upregulated and -downregulated miRNAs showed significantly high and low level expressions, respectively, in glioma mouse models as compared to normal control mice, confirming the reliability of urine miRNA-based diagnosis. Furthermore, some of these glioma-upregulated miRNAs has been reported to be involved in tumor progression. CONCLUSIONS miRNAs obtained from urine could be biomarkers for detection of gliomas by machine learning and some of these could be associated with tumor progression.

Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-\u03baB pathway

BackgroundHepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy.MethodsqRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway.ResultsWe found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-β and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic.ConclusionsOur findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy.