Development Of Metastatic Tumors Research Articles

Abstract A034: 3D in-vitro model picturing cancer-stromal cell crosstalk & bone metastasis

Abstract Breast cancer is presently the most prevalent malignancy and the second leading cause of tumor-related deaths among women. Approximately 75% of patients with advanced breast cancer will develop bone metastasis. Despite the significant improvement in treating primary breast cancer, the 5-year survival rate for metastatic breast cancer remains at 25%. While we recognize the critical importance of cancer cells and Tumor Microenvironment (TME) communication, the field still suffers from a lack of a proper model system to reconstitute 3D multi-cellular TME in-vitro, especially for bone metastasis. Most of the current 3D in-vitro breast cancer models are composed of cancer cells only and are unable to mimic the complex cellular homeostasis within the TME. To address this deficiency along with the tumor heterogeneity, we aimed to develop a unique 3D multi-cellular culture system using patient-derived (PD) cancer cells. In this study, we implemented the “hanging drop” method to create 3D Tumor Spheroids (TSs). This method was not only sufficient to build the 3D structure, but also significantly enhanced patient-derived cancer cell line establishment rate up to 75%. 3D TSs maintained their histopathological characteristics and original hormone receptor status. Over time, TSs exhibited intratumoral hypoxia leading to significant induction of VEGF expression and EMT showcasing the quality of the system. To investigate the complex interrelation between the tumor and TME, we cultured TSs with Endothelial Cells (ECs) and pericytes to introduce the vascular component. To probe these tumor-stromal interactions, we incorporated Cancer-Associated Fibroblasts (CAFs) into TSs and observed the successful growth of Multi-Cellular Tumor Spheroids (MCTSs) alongside the tubular structure. Analysis of our data revealed that the presence of stromal cells in TME enhanced tumor survival. In this study, we define how CAFs aid in the formation of vascular structures of ECs. Recent studies also have found that the effects of CAFs are significantly high in aggressive TNBC and HER2+ cell lines compared to HR+. The long-term goal of this current study is to explore bone-mimicking nano-clay-based 3D scaffold culture systems to investigate the development of metastatic tumors and to understand the influence of bone TME on cellular responses in-vitro. Citation Format: Shrinwanti Ghosh. 3D in-vitro model picturing cancer-stromal cell crosstalk & bone metastasis [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 A034.

Forces at play: exploring factors affecting the cancer metastasis.

Metastatic disease, a leading and lethal indication of deaths associated with tumors, results from the dissemination of metastatic tumor cells from the site of primary origin to a distant organ. Dispersion of metastatic cells during the development of tumors at distant organs leads to failure to comply with conventional treatments, ultimately instigating abrupt tissue homeostasis and organ failure. Increasing evidence indicates that the tumor microenvironment (TME) is a crucial factor in cancer progression and the process of metastatic tumor development at secondary sites. TME comprises several factors contributing to the initiation and progression of the metastatic cascade. Among these, various cell types in TME, such as mesenchymal stem cells (MSCs), lymphatic endothelial cells (LECs), cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), T cells, and tumor-associated macrophages (TAMs), are significant players participating in cancer metastasis. Besides, various other factors, such as extracellular matrix (ECM), gut microbiota, circadian rhythm, and hypoxia, also shape the TME and impact the metastatic cascade. A thorough understanding of the functions of TME components in tumor progression and metastasis is necessary to discover new therapeutic strategies targeting the metastatic tumor cells and TME. Therefore, we reviewed these pivotal TME components and highlighted the background knowledge on how these cell types and disrupted components of TME influence the metastatic cascade and establish the premetastatic niche. This review will help researchers identify these altered components' molecular patterns and design an optimized, targeted therapy to treat solid tumors and restrict metastatic cascade.

Isoprenylcysteine carboxyl methyltransferase (ICMT) promotes invadopodia formation and metastasis in cancer cells

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors.

LncRNA MAGI2-AS3 inhibites tumor progression by up-regulating STAM via interacting with miR-142-3p in clear cell renal cell carcinoma.

Revealing the role of non-coding RNAs (ncRNAs) in inducing dysregulated pathological responses to external signals may identify therapeutic targets for inhibiting the progression of clear cell renal cell carcinoma (ccRCC). Non-coding RNAs belong to a class of RNA molecules that do not encode proteins but possess diverse biological functions, playing essential roles in the occurrence and development of metastatic and proliferative tumors. To investigate the impact of the upstream interaction between miR-142-3p and lncRNA MAGI2-AS3 on the tumor-suppressive activity of the STAM gene, we firstly conducted bioinformatics analysis to predict the upstream miRNAs of STAM and the upstream lncRNAs of the miRNAs through online databases (miRanda, miRDB, TargetScan, LncBase v2), which were further validated by the starBasev2.0 database. Subsequently, multiple experimental techniques were employed to validate these findings, including RT-qPCR, Western blotting, measurement of cellular functional activity, and luciferase reporter assays. Through these experimental methods, we provided compelling evidence regarding the role of miR-142-3p and MAGI2-AS3 in regulating STAM gene expression and functionality, revealing their potential significance in tumor suppression. Our research demonstrates the importance of the MAGI2-AS3/miR-142-3p/STAM signaling pathway axis in ccRCC. MAGI2-AS3 competes for binding with miR-142-3p, resulting in upregulated STAM gene expression. This upregulation inhibits tumor proliferation and metastasis in ccRCC cells. Conversely, overexpression of miR-142-3p or silencing of MAGI2-AS3 promotes tumor behavior, while downregulation of miR-142-3p inhibits the development of ccRCC. Targeting the MAGI2-AS3/miR-142-3p/STAM axis holds promise as a therapeutic strategy for ccRCC treatment.

Combination losartan with hyaluronic acid modified diethyldithiocarbamate loaded hollow copper sulfide nanoparticles for the treatment of breast cancer and metastasis

The application of photothermal therapy (PTT) is greatly limited by the low accumulation of photothermal agents, uneven photothermal distribution, and heat endurance of cancer cells. Worse still, despite PTT enhances immunogenicity, the anti-tumor immune efficacy is still unsatisfactory due to the inefficient immunogenic cell death (ICD) induction and poor infiltration of immune cells. To solve the above problems of PTT, we developed hyaluronic acid (HA) modified hollow copper sulfide nanoparticles encapsulating diethyldithiocarbamate (DDTC) to construct a breast tumor targeting and near infrared (NIR) photo-responsive drug delivery system (D-HCuS@HA), which further combined with losartan to improve the accumulation and penetration in the tumor site. Upon irradiation, D-HCuS@HA realized enhanced PTT and released cytotoxic Cu(DDTC)2 to eliminate heat endurance tumor cells, thereby enhancing anti-tumor effect and inducing effective ICD. Moreover, the combination with losartan could remodel the tumor microenvironment, allowing more T cells to infiltrate into the tumor, and significantly inhibiting the occurrence and development of metastatic tumors. In vitro/vivo results revealed the great potential of D-HCuS@HA combined with losartan, which provides a new paradigm for anti-tumor and anti-metastases.

Cancer stem cells: an insight into the development of metastatic tumors and therapy resistance.

The term "cancer stem cells" (CSCs) refers to cancer cells that exhibit traits parallel to normal stem cells, namely the potential to give rise to every type of cell identified in a tumor microenvironment. It has been found that CSCs usually develops from other neoplastic cells or non-cancerous somatic cells by acquiring stemness and malignant characteristics through particular genetic modifications. A trivial number of CSCs, identified in solid and liquid cancer, can give rise to an entire tumor population with aggressive anticancer drug resistance, metastasis, and invasiveness. Besides, cancer stem cells manipulate their intrinsic and extrinsic features, regulate the metabolic pattern of the cell, adjust efflux-influx efficiency, modulate different signaling pathways, block apoptotic signals, and cause genetic and epigenetic alterations to retain their pluripotency and ability of self-renewal. Notably, to keep the cancer stem cells' ability to become malignant cells, mesenchymal stem cells, tumor-associated fibroblasts, immune cells, etc., interact with one another. Furthermore, CSCs are characterized by the expression of particular molecular markers that carry significant diagnostic and prognostic significance. Because of this, scientific research on CSCs is becoming increasingly imperative, intending to understand the traits and behavior of cancer stem cells and create more potent anticancer therapeutics to fight cancer at the CSC level. In this review, we aimed to elucidate the critical role of CSCs in the onset and spread of cancer and the characteristics of CSCs that promote severe resistance to targeted therapy.

Impact of Obesity on Perioperative Complications on Treatment of Spinal Metastases: A Multicenter Surveillance Study from the German Spine Registry (DWG-Register).

Background The spine is a common location for the development of primary and metastatic tumors, spinal metastases being the most common tumor in the spine. Spinal surgery in obesity is challenging due to difficulties with anesthesia, intravenous access, positioning, and physical access during surgery. The objective was to investigate the effect of obesity on perioperative complications by discharge in patients undergoing surgery for spinal metastases. Methods Retrospective analysis of data from the DWG-register on patients undergoing surgery for metastatic disease in the spine from January 2012 to December 2016. Preoperative variables included obesity (≥ 30 kg/m 2 ), age, gender, and smoking status. In addition, the influence of pre-existing medical comorbidity was determined, using the American Society of Anesthesiologists (ASA) score. Results In total, 528 decompressions with and without instrumentation undergoing tumor debulking, release of the neural structures, or tumor extirpation in metastatic disease of the spine were identified; 143 patients were obese (body mass index [BMI] ≥ 30 kg/m 2 ), and 385 patients had a BMI less than 30 kg/m 2 . The mean age in the group with BMI 30 kg/m 2 or higher (group 1) was 67 years (56.6%). In the group with BMI less than 30 kg/m 2 (group 2), the mean age was 64 years. Most of the patients had preoperatively an ASA score of 3 and 4 (patients with severe general disease). The likelihood of being obese in the logistic regression model seems to be protective by 47.5-fold for blood loss 500 mL or higher. Transfusions occurred in 321/528 (60.7%) patients (group 1, n = 122 and group 2, n = 299; p = 0.04). A total of 19 vertebroplasties with percutaneous stabilization (minimally invasive spine [MIS]), 6 vertebroplasties, and 31 MIS alone were identified. The variables between these groups, with exception of preoperative status (ASA-score; p = 0.02), remained nonsignificant. Conclusion Obese patients were predisposed to have blood loss more than 500 mL more often than nonobese patients undergoing surgery for spinal metastases but with perioperative blood transfusions, invasiveness, nor prolonged hospitalization. Early postoperative mobilization and a low threshold for perioperative venous thromboembolism (VTE) are important in obese patients to appropriately diagnose, treat complications, and minimize morbidity.

Metformin and histone deacetylase inhibitor based anti-inflammatory nanoplatform for epithelial-mesenchymal transition suppression and metastatic tumor treatment

Epithelial-mesenchymal transition (EMT), a differentiation process with aberrant changes of tumor cells, is identified as an initial and vital procedure for metastatic processes. Inflammation is a significant inducer of EMT and provides an indispensable target for blocking EMT, however, an anti-inflammatory therapeutic with highlighted safety and efficacy is deficient. Metformin is a promising anti-inflammatory agent with low side effects, but tumor monotherapy with an anti-inflammation drug could generate therapy resistance, cell adaptation or even promote tumor development. Combination therapies with various anti-inflammatory mechanisms can be favorable options improving therapeutic effects of metformin, here we develop a tumor targeting hybrid micelle based on metformin and a histone deacetylase inhibitor propofol-docosahexaenoic acid for efficient therapeutic efficacies of anti-inflammatory drugs. Triptolide is further encapsulated in hybrid micelles for orthotopic tumor therapies. The final multifunctional nanoplatforms (HAOPTs) with hyaluronic acid (HA) modification can target tumor efficiently, inhibit tumor cell EMT processes, repress metastasis establishment and suppress metastatic tumor development in a synergistic manner. Collectively, the results afford proof of concept that the tumor targeting anti-inflammatory nanoplatform can provide a potent, safe and clinical translational approach for EMT inhibition and metastatic tumor therapy.

Abstract 3045: Distinct oral microbial signatures are associated with primary and metastatic brain tumors

Abstract Background: The role of microbiome in different steps of tumor development and progression has been shown across various tumor types. However, the contribution of microbiome to brain tumors is yet to be understood. A crosstalk between the gut and oral microbiota and the brain microenvironment has been demonstrated in the context of neurological and neurodegenerative diseases. We hypothesize that distinct gut and oral microbial signatures are associated with primary and metastatic brain tumors. Methods: Matched stool, saliva, and buccal swab samples were collected prospectively from patients with primary or metastatic brain tumors who underwent surgical tumor resection at the University of Texas MD Anderson Cancer Center. Samples were collected using the OMNIgene microbiome collection and stabilization kits (DNAgenotek, Kit Number OM-200, OMR-120, and OM-505 for stool, buccal swab, and saliva, respectively). Samples were then processed, aliquoted, and stored at -80 C. Samples from 10 patients for each disease type were sequenced via metagenomic shotgun sequencing. Taxonomic profiling was conducted through MetaPhlAn and Virmap, for bacterial and viral taxa, respectively. Beta diversity was analyzed using Bray-Curtis and UniFrac analyses. Biom files were further analyzed to identify the signatures associated with each disease type, using the Phyloseq and Microbiome packages in R and heatmaps were generated using the Pheatmap package. Results: Microbiome beta diversity analysis demonstrated distinct clustering of stool, saliva, and buccal swab samples in both primary and metastatic brain tumor patients, which confirmed high sample quality and lack of cross-contamination between buccal swab and oral samples during sample collection. Analysis of oral microbiome samples demonstrated distinct bacterial and viral signatures that were enriched in metastatic brain tumors in comparison to primary brain tumors. In contrast, the composition of gut microbial taxa was comparable between metastatic and primary brain tumors and demonstrated enrichment in Bacteroides and Microviridae families. Conclusion: Our analysis of gut and oral microbiome in patients with primary and metastatic brain tumors suggest that distinct bacterial and viral taxa within the oral microbiome are enriched in metastatic brain tumors. We are in the process of expanding our clinical cohort to further validate these findings. Moreover, mechanistic studies are ongoing to understand the contribution of microbial signatures to the development and progression of metastatic and primary tumors. (These studies were supported by the National Institute of Health (F32CA260769) and the Glioblastoma Moonshot Program at the MD Anderson Cancer Center.) Citation Format: Golnaz Morad, Matthew Lastrapes, Matthew Wong, Pranoti Sahasrabhojane, Sherise Ferguson, Nadim Ajami, Jennifer Wargo. Distinct oral microbial signatures are associated with primary and metastatic brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3045.

Abstract 3295: The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology

Abstract Nucleic acid drugs have shown promise in oncology, however their use is limited by hepatic clearance. Due to the mononuclear phagocytic system (MPS), a large part of the intravenously-administered dose does not reach the therapeutic target. Our approach aims to address this issue using biocompatible nanoparticles—the Nanoprimer—that is administered prior to the therapeutic. Due to its physico-chemical properties, the Nanoprimer is transiently internalized by Kupffer cells (KC), and endothelial cells of the MPS, enabling temporary reduction of hepatic clearance. These data further explore the Nanoprimer’s mechanism of action and demonstrate that scavenger receptor-A (SR) is the main receptor involved in Nanoprimer binding and uptake by KC. Efficacy studies in mouse model show that the Nanoprimer dramatically increases the anti-tumor efficacy of nucleic acid-based treatment without introducing additional toxicities. An in vitro toxicity evaluation was performed investigating the Nanoprimer’s hemolytic properties and its potential ability to activate the complement system, platelet aggregation and pro-inflammatory cytokines*. SR-mediated binding and uptake of Nanoprimer by KC were assessed using a competitive binding assay with SR inhibitor polyinosinic acid. The impact of the Nanoprimer on anti-tumor efficacy was evaluated on orthotopic triple negative breast cancer (TNBC) model in combination with siRNA-loaded lipid nanoparticles (LNPs) targeting a novel cancer-specific gene transcript of a master regulator of invadopodia formation and Wnt, Hedgehog and Notch oncogenic pathways. Results showed that the Nanoprimer is safe at the effective dose. The Nanoprimer does not activate the complement components, induce hemolysis, platelet aggregation, or cytokine storm syndrome elements. The competitive inhibition of SR-A decreases Nanoprimer binding and KC-uptake by 70%, indicating SR-A selective uptake of the Nanoprimer by macrophages. The efficacy study shows that combining the Nanoprimer with siRNA LNP therapy significantly increased treatment efficiency, with a mean tumor growth inhibition ratio of 36.3% for LNPs alone versus 84.6% for the combination. Moreover, the combination therapy more efficiently prevents the development of pulmonary metastatic tumors, with a metastasis-inhibition rate of 98%. These data demonstrate the safety of the Nanoprimer, along with its ability to maximize the inhibition of primary tumor growth and pulmonary metastasis by systemic siRNA LNP therapy in a TNBC mouse model. They also highlight the capacity of Nanoprimer technology to unlock the potential of nucleic acid-based treatments. The ubiquitous nature of its mode of action allows its application to a broad spectrum of anti-cancer therapeutics. *we acknowledge NCL, Frederick National Laboratory for Cancer Research sponsored by the NCI for the help with conducting these experiments Citation Format: Julie Devallière, Laurence Poul, Maxime Bergère, Audrey Darmon, Océane Jibault, Francis Mpambani, Kelvin Tsai, Matthieu Germain. The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3295.

Abstract 3451: Microglial phagocytosis suppressed the development of metastatic tumors in the imaging model of brain metastasis

Abstract Brain metastasis of lung cancer is a crucial problem that causes poor clinical outcomes and alters patient quality of life. Previous evidence showed that lung cancer cells (LUCs) may escape from host immunity to form distant metastases. We previously used an original imaging model of brain metastasis (IBrM) to show a heterogeneous interaction between microglia, the tissue macrophages in the brain, and LUCs in the brain niche. The findings confirmed that microglia can phagocytose metastatic cancer cells. Here, we show that genetic deletion of the “don’t eat me” signals of cancer cells can promote phagocytic activity of microglia against LUCs. We injected an adenocarcinoma lung cancer cell line expressing mCherry (CMT167mC) via the internal carotid artery of the CX3CR1-EGFP mice, whose microglia had been specifically labeled with EGFP. The microglia and LUCs were visualized in vivo simultaneously by two-photon microscopy for 14 days. The tumor fate and microglial response against cancer cells were evaluated at single cell resolution in the IBrM. CD24, CD47, and/or PDL1 were deleted in CMT167mC cells using CRISPR/Cas9 to examine the effect of the “don’t eat me” signals. The dynamic behavior of LUCs and the microglial phagocytosis activity against LUCs were visualized in living mice. Microglial phagocytosis was significantly increased by single deletion of CD47 or CD24 compared with WT cells. Combined deletion of CD47 and CD24 also synergically increased microglial phagocytosis, resulting in reduced IBrM and prolonged mouse survival. By contrast, deletion of PD-L1 did not enhance microglial phagocytosis or prolong survival. The pharmacological or genetic conditional depletion of microglia canceled both the increased phagocytosis and the extended survival time, suggesting that the effect of “don't eat me” signals was microglia-dependent. These results indicate that suppression of both CD24 and CD47 in LUCs enhances microglial phagocytic activity and suppresses the metastatic formation of LUCs. Furthermore, the combined use of CRISPR/Cas9 in LUCs and in vivo imaging allows us to evaluate potential therapeutic targets associated with microglial phagocytosis. We are currently using single cell RNA-seq and cell-to-cell communication analysis of the brain tumor and surrounding niche to explore other signal pathways that regulate tumor phagocytosis by microglia and to identify potential therapeutic targets. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Microglial phagocytosis suppressed the development of metastatic tumors in the imaging model of brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3451.

The Role of the Innate Immune System in Cancer Dormancy and Relapse.

Simple SummaryOverall survival of patients with cancer is dependent on the success of therapy. Therapy failure is correlated with enhanced metastasis and recurrence of the primary tumor. However, metastases may develop before the detection of a primary tumor and become dormant at their secondary site, presenting a major clinical challenge as these dormant cells can reactivate after the completion of seemingly successful therapy. Research has demonstrated that the innate immune system plays an integral role in molecular crosstalk with cancer cells to facilitate metastatic dissemination and control over a dormant cell state. Here, we discuss which types of innate immune cells are engaged in this crosstalk at each stage of the metastatic cascade. We also highlight how different subtypes of innate immune cells induce dormancy in cancer cells and facilitate the emergence from a dormant state. Lastly, we examine current therapeutic strategies aimed at inhibiting immune-mediated metastasis and dormancy.Metastatic spread and recurrence are intimately linked to therapy failure, which remains an overarching clinical challenge for patients with cancer. Cancer cells often disseminate early in the disease process and can remain dormant for years or decades before re-emerging as metastatic disease, often after successful treatment. The interactions of dormant cancer cells and their metastatic niche, comprised of various stromal and immune cells, can determine the length of time that cancer cells remain dormant, as well as when they reactivate. New studies are defining how innate immune cells in the primary tumor may be corrupted to help facilitate many aspects of dissemination and re-emergence from a dormant state. Although the scientific literature has partially shed light on the drivers of immune escape in cancer, the specific mechanisms regulating metastasis and dormancy in the context of anti-tumor immunity are still mostly unknown. This review follows the journey of metastatic cells from dissemination to dormancy and the onset of metastatic outgrowth and recurrent tumor development, with emphasis on the role of the innate immune system. To this end, further research identifying how immune cells interact with cancer cells at each step of cancer progression will pave the way for new therapies that target the reactivation of dormant cancer cells into recurrent, metastatic cancers.

S3611 Double Trouble: Primary Lung and Colon Cancer in a Patient With Crohn’s Disease

Introduction: This vignette highlights the development of localized rectal adenocarcinoma and concomitant metastatic large cell neuroendocrine tumor of the lung in a patient with longstanding Crohn’s disease (CD) on Vedolizumab (VDZ). Case Description/Methods: A 61 year old male with a 2 year history of severe, uncontrolled colonic inflammatory CD presented to the ED with weakness, loss of appetite, and a 30lb weight loss over a 1 month period. He had been on VDZ 300mg every 8 weeks for 1 year. A CT chest showed a new 2cm pulmonary nodule with increasing mediastinal and perihilar lymphadenopathy. Bronchoscopy and endobronchial ultrasound were performed for lymph node sampling and revealed a large cell neuroendocrine tumor, which was later found to be metastatic to the brain. The patient was discharged with plans to optimize the VDZ therapy and start cancer treatment. Anticipating the use of immunotherapy, a colonoscopy was performed and revealed active inflammation in the sigmoid colon. Incidentally, an 18mm Is (sessile) polyp was found in the rectum and removed by piecemeal snare polypectomy. Pathology was consistent with invasive colonic adenocarcinoma. A colonoscopy one year earlier had been negative for malignancy. After a careful discussion in the GI tumor board, the decision was made to treat the lung cancer first. Unfortunately, the patient developed sepsis after his first chemotherapy dose and passed away. Discussion: This case illustrates the challenges with CD therapy during active malignancy. CD patients are at increased risk for both colonic adenocarcinoma and pulmonary malignancy. There have been isolated cases linking CD to premalignant diffuse idiopathic pulmonary neuroendocrine cell hyperplasia, suggesting a possible correlation between CD and the highly aggressive large cell neuroendocrine tumor of the lung seen, though data is limited. VDZ has been associated with 24%-31% clinical remission of CD after induction. It was shown to be well tolerated and had a low risk of malignancy in patients with and without a history of prior malignancy. Data is limited on the use of VDZ concurrently with active chemotherapy, though it is presumed have a favorable safety profile given the gut-selective nature of the drug. The development of malignancies in this case is thought to be possibly related to CD and likely unrelated to VDZ. With limited data available, a multidisciplinary discussion between oncology and GI is ideal to develop a tailored treatment plan for CD patients with active malignancy.

PHLDA3 Is an Important Downstream Mediator of p53 in Squamous Cell Carcinogenesis

Squamous cell carcinomas (SCCs) are one of the most frequent solid cancer types in humans and are derived from stratified epithelial cells found in various organs. SCCs derived from various organs share common important properties, including genomic abnormalities in the tumor suppressor gene p53. There is a carcinogen-induced mouse model of SCC that produces benign papilloma, some of which progress to advanced carcinoma and metastatic SCCs. These SCCs undergo key genetic alterations that are conserved between humans and mice, including alterations in the genomic p53 sequence, and are therefore an ideal system to study the mechanisms of SCC tumorigenesis. Using this SCC model, we show that the PHLDA3 gene, a p53-target gene encoding a protein kinase B repressor, is involved in the suppression of benign and metastatic tumor development. Loss of PHLDA3 induces an epithelial‒mesenchymal transition and can complement p53 loss in the formation of metastatic tumors. We also show that in human patients with SCC, low PHLDA3 expression is associated with a poorer prognosis. Collectively, this study identifies PHLDA3 as an important downstream molecule of p53 involved in SCC development and progression.

Bone Morphogenic Protein Signaling and Melanoma.

Malignant melanoma is a deadly form of skin cancer caused by neoplastic transformation of melanocytic cells. Despite recent progress in melanoma therapy, by inhibition of activated oncogenes or immunotherapy, survival rate for metastatic melanoma patients remains low. The remarkable phenotypic plasticity of melanoma cells allows for rapid development of invasive properties and metastatic tumors, the main cause of mortality in melanoma patients. Phenotypic and molecular analyses of developing tumors revealed that epithelial-mesenchymal transition (EMT), a cellular and molecular mechanism, controls transition from mature melanocyte to less differentiated melanocyte lineage progenitor cells forming melanoma tumors. This transition is facilitated by persistence of transcriptional regulatory circuit characteristic of embryonic stage in mature melanocytes. Switching of the developmental program of mature melanocyte to EMT is induced by accumulated mutations, especially targeting BRAF, N-RAS, or MEK1/2 signaling pathways, and further promoted by dynamic stimuli from local environment including hypoxia, interactions with extracellular matrix and growth factors or cytokines. Recent reports demonstrate that signaling mediated by transforming growth factor-β (TGF-β) and bone morphogenic proteins (BMPs) play critical roles in inducing EMT by controlling expression of critical transcription factors. BMPs are essential modulators of differentiation, proliferation, apoptosis, invasiveness, and metastases in developing melanoma tumors. They control transcription and epigenetic landscape of melanoma cells. Better understanding of the role of BMPs may lead to new strategies to control EMT processes in melanocyte cell lineage and to achieve clinical benefits for the patients.

Role of platelets and breast cancer stem cells in metastasis.

The high mortality rate of breast cancer is mainly caused by the metastatic ability of cancer cells, resistance to chemotherapy and radiotherapy, and tumor regression capacity. In recent years, it has been shown that the presence of breast cancer stem cells is closely associated with the migration and metastatic ability of cancer cells, as well as with their resistance to chemotherapy and radiotherapy. The tumor microenvironment is one of the main molecular factors involved in cancer and metastatic processes development, in this sense it is interesting to study the role of platelets, one of the main communicator cells in the human body which are activated by the signals they receive from the microenvironment and can generate more than one response. Platelets can ingest and release RNA, proteins, cytokines and growth factors. After the platelets interact with the tumor microenvironment, they are called "tumor-educated platelets." Tumor-educated platelets transport material from the tumor microenvironment to sites adjacent to the tumor, thus helping to create microenvironments conducive for the development of primary and metastatic tumors. It has been observed that the clone capable of carrying out the metastatic process is a cancer cell with stem cell characteristics. Cancer stem cells go through a series of processes, including epithelial-mesenchymal transition, intravasation into blood vessels, movement through blood vessels, extravasation at the site of the establishment of a metastatic focus, and site colonization. Tumor-educated platelets support all these processes.

Pericyte-secreted IGF2 promotes breast cancer brain metastasis formation.

Brain metastases are life‐threatening complications of triple‐negative breast cancer, melanoma, and a few other tumor types. Poor outcome of cerebral secondary tumors largely depends on the microenvironment formed by cells of the neurovascular unit, among which pericytes are the least characterized. By using in vivo and in vitro techniques and human samples, here we show that pericytes play crucial role in the development of metastatic brain tumors by directly influencing key steps of the development of the disease. Brain pericytes had a prompt chemoattractant effect on breast cancer cells and established direct contacts with them. By secreting high amounts of extracellular matrix proteins, pericytes enhanced adhesion of both melanoma and triple‐negative cancer cells, which might be particularly important in the exclusive perivascular growth of these tumor cells. In addition, pericytes secreted insulin‐like growth factor 2 (IGF2), which had a very significant pro‐proliferative effect on mammary carcinoma, but not on melanoma cells. By inhibiting IGF2 signaling using silencing or picropodophyllin (PPP), we could block the proliferation‐increasing effect of pericytes on breast cancer cells. Administration of PPP (a blood–brain barrier‐permeable substance) significantly decreased the size of brain tumors in mice inoculated with triple‐negative breast cancer cells. Taken together, our results indicate that brain pericytes have significant pro‐metastatic features, especially in breast cancer. Our study underlines the importance of targeting pericytes and the IGF axis as potential strategies in brain metastatic diseases.

Effect of general anesthetics on the tumor micro-environment in mouse models of breast cancers of spontaneous metastasis.

e15503 Background: Metastatic breast cancer is a pressing health concern worldwide. Various treatments have been developed but no significant long-term changes in overall survival are observed. Therefore, there is a demand to improve current therapies to treat this disease. Surgical resection of the primary tumors is essential in the treatment. However, accumulating evidence alludes to a role for volatile anesthetics which are used during the surgery in metastatic tumor development, but the mechanism remains largely unknown. We have shown anesthetics exert different effects on lung metastasis in mouse models of breast cancers. This study analyses the effect of general anesthetics in lung microenvironment associated with the increased metastases. Methods: Balb/c mice and NOD-SCID mice were orthotopically implanted with 4T1 cells and MDA-MB-231 cells respectively, in the mammary fat pad to generate primary tumors. Mice were subjected to the tested anesthetic during implantation and/or before and after surgery. Surgical dissection of primary tumor was performed under anesthesia with sevoflurane or an intravenous anesthetic propofol. Survival curve was constructed and analysed. Mice were euthanized to harvest tissues for histology and cell analysis. Results: As we previously reported, surgical dissection of primary tumor in mice under anesthesia with sevoflurane led to significantly more lung metastasis than with propofol in both syngeneic murine 4T1 and xenograft human MDA-MB-231 breast cancer models. Sevoflurane was associated with increased IL6(Li, Huang, & Lin, 2020). Here we show that anesthesia with sevoflurane resulted in changes of stroma composition in the lung, which was reversed by IL6 pathway interruption. Conclusions: Those results contribute to our understanding of effects of sevoflurane on cancer metastasis and suggest a potential therapeutic approach to overcome the risk of general anesthesia. Li, R., Huang, Y., & Lin, J. (2020). Distinct effects of general anesthetics on lung metastasis mediated by IL-6/JAK/STAT3 pathway in mouse models. Nat Commun, 11, 642.

An AAV6-based multi-antigen vaccine for treatment of murine melanoma

Abstract We recently showed that a vaccine with optimized AAV6 vector generates a superior immune response against strong antigens (Ag) such as Ovalbumin. However, tumor-associated antigens (TAA) are typically non-mutated self-proteins with low immunogenicity. To increase vaccine efficacy and to reduce tumor evasion of immune attack, we developed a multivalent AAV vaccine that targets four previously characterized melanoma TAA: tyrosinase, premelanosome protein gp100, tyrosinase-related protein 1, and dopachrome tautomerase. The vaccine represents a combination of individual AAV-based vaccines specific to unique TAA. The strength of cytotoxic T cell response against each TAA was shown via IFNγ ELISPOT analysis of re-stimulated splenocytes from immunized mice. Additionally, we showed that vaccine-induced Ag-specific antibodies directed B16F10 melanoma cells to complement-dependent lysis. The vaccine’s therapeutic potential was analyzed in prevention and treatment of B16F10 mouse melanoma. The preventative vaccination significantly reduced the number of metastatic tumor nodules in lungs. Since B16F10 melanomas are immunologically resistant tumors, we combined vaccination with aPD-1 and aPD-L1 checkpoint inhibitors for treatment of established solid tumor. The combination of both vaccine and checkpoint inhibitors significantly delayed tumor growth and extended animal survival. In summary, our preliminary data suggest that a multivalent vaccine based on modified AAV6 vectors generates strong immune responses against endogenous self-proteins, and vaccination may have a therapeutic benefit against both metastatic and solid tumor development.

Cancer stemness contributes to cluster formation of colon cancer cells and high metastatic potentials.

The ability of cancer cells to form clusters is a characteristic feature in the development of metastatic tumours with drug resistance. Several studies demonstrated that clusters of circulating tumour cells (CTCs) have a greater metastatic potential to establish new tumours at secondary sites than single CTCs. However, the mechanism of cluster formation is not well understood. In this study, we investigated whether cancer stemness would contribute to cluster formation. We used a tumour sphere culture method to enrich cancer stem cells (CSCs) from colon cancer cells and found that during the second generation of sphere culture, clusters (between 3 and 5 cells) formed within the first 24hours, whereas the rest remained as single cells. The clusters were analysed for stemness and metastatic potential, including gene expressions for cancer stemness (CD133 and Lgr5), epithelial-mesenchymal transition (E-cadherin and TGF-β 1-3) and hypoxia-induced factors (HIF-1α and HIF-2α). The results showed that the clusters expressed higher levels of these genes and colon CSC surface markers (including CD24, CD44 and CD133) than the single cells. Among these markers, CD24 seemed the major contributor linking the cells into the clusters. These clusters also showed a stronger ability to both form colonies and migrate. Our data collectively suggest that colon cancer stemness contributes to cluster formation and that clustered cells exhibit a great metastatic potential. Our study thus provides a method to study the CTC clusters and derive insight into oncogenesis and metastasis.