Pre-metastatic Niche Research Articles

Abstract 6000: A millifluidic culture platform for quantifying premetastatic niche cultivation in quiescent organ stroma induced by real-time transport of tumor-derived factors

Abstract Progression to metastasis contributes to the poor overall survival of patients diagnosed with aggressive cancers such as triple negative breast cancer (TNBC). Tumor-derived factors (TDF) induce pathological changes in distant organ niches that render these organs more receptive to colonization by disseminated tumor cells. This process, termed premetastatic niche (PMN) cultivation, is characterized in part by pathological activation of stromal cells and induction acute extracellular matrix (ECM) deposition. There is an urgent and unmet need for models of PMN cultivation in human cancers to accelerate the discovery of mechanisms and develop PMN-targeting therapies. We engineered 3D culture models of aggressive carcinoma and quiescent organ interstitium and integrated them in a millifluidic device that enables interconnected coculture. Our devices are engineered for ease of adoption by labs with experience culturing cells and organoids in 3D hydrogels. Cultures can be performed in static, with continuous gravity-driven flow, or with a rocker for recirculating flow. Here we present a form of the model in which a patient-derived triple negative breast cancer microenvironment is linked with a quiescent lung interstitium. Fibroblast activation and ECM synthesis indicative of PMN cultivation are quantified relative to a physiological resting state. All device cultures were maintained with fibroblast growth medium containing 0.1% FBS and 1% ITS which maintains the lung compartment in a quiescent state in the absence of endogenous activating factors in the system. The TME compartment in our devices exhibited features of aggressive cancers after 7 days of culture including widespread 3D invasion of tumor cells and formation of a dense stroma comprised of myofibroblast-like CAF with prominent smooth muscle actin (SMA) cytoskeleton and nuclear translocation of activated Yes-associated protein 1 (aYAP1). These cellular features of the engineered tumor microenvironment were accompanied by a dense anisotropic matrix of newly-synthesized fibronectin. TNBC-derived factors transported in the system induced PMN cultivation in the lung compartment by 7 days, as evidenced by quantitative increases in focal deposition of fibronectin and a spectrum of activated phenotypes defined by SMA and aYAP1 expression patterns in previously quiescent fibroblasts. Thus, we have captured cancer-induced changes in the density and architecture of newly-synthesized ECM in a distant tissue that would support tumor cell adhesion. Citation Format: Elisabet A. Olsen, Omar M. Ahmad, Bradley Sweeney, Caroline C. Culp, Yasmin V. Maurice, Matthew R. Burow, Mark Mondrinos. A millifluidic culture platform for quantifying premetastatic niche cultivation in quiescent organ stroma induced by real-time transport of tumor-derived factors [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 6000.

Abstract 2190: Brain-seeking extracellular vesicles derived from metastatic breast cancer cells modulate brain endothelial cell metabolism

Abstract Breast cancer, particularly the triple-negative subtype, is the primary cause of brain metastasis in women and affects ~10-16% of breast cancer patients. Breast cancer patients diagnosed with brain metastasis have limited therapeutic options and, therefore, a dismal prognosis with a median survival time of ~10 months. Recent studies have suggested that metastatic cells extravasate and grow along the blood-brain barrier (BBB) in a favorable microenvironment called the pre-metastatic niche. However, the early steps leading to the onset of breast-to-brain metastasis are poorly understood, limiting the development of strategies to prevent, detect and treat premetastatic or early-stage lesions. It is now well accepted that extracellular vesicles (EVs), nanoparticles secreted from cancer cells, can travel to distant organs, activate resident cells and promote the remodeling of the extracellular matrix and significantly impact organ-specific metastatic spread. Using our mouse model of breast to brain metastasis, we have demonstrated, for the first time, that EVs derived from brain-seeking breast cancer cells (Br-EVs) breach the intact BBB via transcytosis, are taken up by astrocytes and cause a significantly increased incidence of brain metastasis. Within this context, we are studying the interactions between Br-EVs and brain endothelial cells (ECs), the latter being a key functional component of the BBB. Utilizing primary human ECs and using a transwell assay established in our laboratory as an in vitro model of the BBB, we have identified the mechanism(s) through which Br-EVs modulate EC endocytic metabolism in favor of increased vesicle recycling and transcytosis. Our mechanistic studies demonstrate that ECs treated with Br-EVs show a significant decrease of multiple target proteins, all of which are involved in the long-recycling loop of the endocytic vesicles. Br-EVs mediate, at least in part, their effect through the delivery to the target cells of a specific subset of micro RNAs. As a result, Br-EVs upregulate EC basal transcytosis rate promoting their transfer to the brain parenchyma. These findings indicate that Br-EVs can alter BBB basal metabolism and help to prepare a microenvironment, better known as the pre-metastatic niche, that promotes and sustains the survival and growth of breast cancer metastases in the otherwise hostile brain microenvironment. These results increase our understanding of the early events that facilitate breast-to-brain metastasis and have the potential to identify key regulators in the early steps of brain metastasis formation. Our mechanistic studies will also inform and contribute to the development of EV-based diagnostic and therapeutic strategies for the early management of breast cancer patients with brain metastasis. (This work was supported by NIH R21 CA253051-01 and the Breast Cancer Research Foundation). Citation Format: Sara Busatto, Golnaz Morad, Marsha Moses. Brain-seeking extracellular vesicles derived from metastatic breast cancer cells modulate brain endothelial cell metabolism [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 2190.

Abstract 5817: Exosomal miR-4466 from nicotine-activated neutrophils promotes tumor cell stemness & metabolism in brain metastasis

Abstract Smoking is associated with lung cancer and has a profound impact on tumor immunity. Nicotine, the addictive and non-carcinogenic smoke component, influences various brain cells and the immune system. However, little is known how long-term use of nicotine affects clinical outcomes in patients with brain metastasis. To address this question, we conducted a retrospective analysis of 810 lung cancer patients with smoking history and assessed brain metastasis. We found that lung cancer patients of current smokers have significantly higher brain metastatic incidence compared to the never-smokers. Immune profiling of the brain metastatic lesions among smokers showed predominant infiltration of neutrophils compared to never-smokers. Notably, mice pre-exposed to nicotine showed a significantly increased number of neutrophils in the brains even before implantation of cancer cells (pre-metastatic niche), and their numbers progressively increased during the metastatic stage. Neutrophil depletion during metastatic colonization caused a remarkable decrease in brain metastasis. Furthermore, we found that chronic exposure of nicotine recruited STAT3-activated N2-neutrophils that secreted exosomal miR-4466 in the brain. This pre-metastatic niche promoted stemness and metabolic switching via SKI/SOX2/CPT1A axis in the tumor cells thereby enabling brain metastasis. Importantly, exosomal miR-4466 levels were found to be elevated in serum/urine of cancer-free subjects with a smoking history and promote tumor growth in vivo, suggesting that exosomal miR-4466 may serve as a promising prognostic biomarker for predicting increased risk of metastatic disease among smoker(s). Our findings suggest a novel pro-metastatic role of nicotine-induced N2-neutrophils in the progression of brain metastasis. We also demonstrated that inhibiting nicotine-induced STAT3-mediated neutrophil polarization effectively abrogated brain metastasis in vivo. Our finding for the first time provides compelling evidence related to the mechanism(s) of neutrophil-mediated brain metastasis that arises from nicotine use. Our results also identified a potential prognostic biomarker for the onset of metastasis as a risk-reduction measure in lung cancer patients and implicated the risk of using nicotine gateway for smoking cessation in cancer patients with a smoking history. Citation Format: Abhishek Tyagi, Shih-Ying Wu, Sambad Sharma, Kerui Wu, Dan Zhao, Ravindra Deshpande, Kounosuke Watabe. Exosomal miR-4466 from nicotine-activated neutrophils promotes tumor cell stemness & metabolism in 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 5817.

Abstract 2223: Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [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 2223.

Abstract 2643: Design and discovery of novel oral INV-88 macrophage migration inhibitory factor (MIF) inhibitors: Potent anti-tumor activity in vitro and in vivo

Abstract Background: MIF is a pleiotropic cytokine critically implicated in tumorigenesis. Tumor cell- and immune effector cell-derived MIF are localized to exosomal, extracellular, cytoplasmic and intranuclear compartments. MIF activates MAPK and PI3K signal transduction, inhibits apoptosis, promotes angiogenesis, elicits premetastatic niche formation and resolves DNA replication stress; contributing to unperturbed cancer cell growth and metastasis. MIF overexpression in several cancers is a negative prognostic indicator associated with poor overall patient survival. Methods & Results: Active novel INV-88 MIF inhibitors were discovered using structure-based drug design against critical MIF pharmacophoric binding sites guided by high-resolution X-ray crystallography (Brookhaven) of INV-88 compound:hu-MIF protein bound complexes. Following an iterative medicinal chemistry campaign; stringent biochemical, pharmacological and ADMET funnel screening identified promising INV-88 lead candidates with potent antitumor activities. Strong MIF binding by INV-88 compounds was demonstrated using thermal shift and fluorescence polarization assays. Profound in vitro antitumor pharmacological properties of INV-88 were established on several murine & human tumor cell lines (HCT116, MeWo, DU145, 4T1, B16-F10, CT26). INV-88 inhibitors demonstrated potent anti-proliferative effects down to nM IC50 potencies and impeded 3D spheroid growth & viability. Disruption of MIF:CD74/CD44-mediated MAPK and PI3K signal transduction pathways by INV-88 compounds blocked ERK and AKT phosphorylation leading to decreased pERK1/2 and pAKT levels. INV-88 inhibitors increased Caspase-3/7, p53 and Bax but decreased Bcl-2 levels. INV-88 lead compounds inhibited tumor MIF protein production in 2D & 3D cell culture systems with concomitant pan-inhibition of tumorigenic proinflammatory cytokine (TNF-α, IL-1β, IL-6, and IL-8) expression by stromal cells. Optimal ADMET properties of INV-88 compounds enabled oral dosing of select INV-88 clinical candidate compounds in mouse syngeneic B16-F10 melanoma and CT26 colon carcinoma models. Mice were dosed daily with INV-88 or vehicle per oral gavage starting in BALB/c and in C57/BL6 mice upon demonstrating palpable tumors after s.c. implantation of CT26 and B16-F10 tumor cells, respectively. Tumor growth inhibition was observed with rates of 71% on Day 24 (CT26) and 75% on Day 20 (B16-F10). INV-88 treatment significantly inhibited CT26 [p<0.0001] and B16-F10 tumor growth [p<0.0001] versus controls. INV-88 was well-tolerated by tumor-bearing mice which exhibited normal body condition scores and body weight. Conclusion: The data presented highlight the potential therapeutic utility of pharmacological MIF inhibition with oral INV-88 new chemical entities as a novel immunotherapeutic treatment modality for cancer. Citation Format: Anderson Gaweco, Vincent Chung, Jefferson Tilley, William Windsor, Terry Stouch, John Ellingboe, Gregg Crichlow, Philipp Sabanas, Christian Sebesta. Design and discovery of novel oral INV-88 macrophage migration inhibitory factor (MIF) inhibitors: Potent anti-tumor activity in vitro and in vivo [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 2643.

Abstract 1581: Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC)

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, which could be attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for developing effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are small extracellular vesicles (sEVs) of approximately 100-150 nm in diameter, often referred as exosomes. Previous report revealed that increased levels of exosomes in NSCLC patients correlated with lower overall survival. Indeed, secretion of sEVs by cancer cells can induce malignant transformation of non-cancerous cells nearby or at distant locations through influencing pre-metastatic niche formation. Therefore, biogenesis and release of cancer derived sEVs are determinant steps for the development of tumor metastasis. However, the mechanisms through which sEVs are produced and secreted by cancer cells are yet to be determined. Here, we isolated and characterized sEVs from a panel of NSCLC cell lines (H358, Calu6, H460, SKMES-1) and non-tumorigenic cells (BEAS-2B and HBEC) to identify mechanisms involved in the elevated secretion of EVs by cancer cells. Contrary to what has been previously reported in literature, our data demonstrated that not all cancer cell lines release high levels of sEVs. Our data suggests that elevated secretion of sEVs could be driven by specific mutations and oncogenic signaling in cancer cells. Among all the cell lines tested, H358 cells which harbor the infamous G12C mutation in KRAS, had the highest sEV release. Therefore, we hypothesize that the KRASG12C can drive high sEV production and secretion in H358 cells. Our preliminary data suggests that siRNA mediated knockdown of oncogenic KRAS signaling in H358 cells significantly decreases secretion of sEVs. Ongoing studies aim to pinpoint the detailed mechanisms by which cancer cells utilize KRAS oncogenic signaling to promote EV biogenesis pathways and tumorigenesis. The current study will provide the foundation to developed new therapeutic strategies against tumor sEVs-driven metastasis. Citation Format: Zulaida M. Soto Vargas, Ikjot Singh Sohal, Humna Hasan, David Arteaga, Andrea L. Kasinski. Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC) [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 1581.

Abstract 3189: Characterization of secretory cues that promote brain metastasis using a microfluidic blood brain niche (BBN) device

Abstract Brain metastases occur in ~ 20% of cancers and are one of the most lethal progression events. The dynamic processes underpinning cancer/tumor microenvironment (TME) interactions are poorly understood. We examined the TME remodeling in response to secretory cues provided by the brain niche components that attract metastatic cancer cells. We identified individual secretions by cell type in the niche. Dkk1 was regulated in response to the brain niche cells exposure to cancer cells; thus, we delineated how Dkk1 modulates the remodeling of the niche and migratory behavior of the cancer cells. We utilized two in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the BBB and brain TME; 2) a migration chip that assesses linear cell migration. The BBN chip consists of two-chambers separated by a porous membrane. The bottom is filled with human astrocytes or their secretions in collagen to recapitulate the pre-metastatic niche. Endothelial cells (hCMEC/D3) form a barrier on the membrane separating the two chambers. MDA-231-BR extravasate into BBN chips containing astrocytes within 2-Days, develop into micro-metastases after 9-Days, and establish cancer – astrocyte contacts. Providing astrocytic secretions in BBN chips produced similar results. Increased interaction was found between brain seeking clones of MDA-MB-231 and JIMT-1 cells and astrocytes compared to parental cells. Analysis of astrocytic and endothelial secretions detected, amongst others, Dkk-1, that is preferentially elevated in astrocytes when stimulated with MDA-231-BR compared to MDA-231. The migration chip was utilized to assess the impact of Dkk-1 on cancer cell migration. Dkk-1 gradients are established using passive diffusion, then migration is monitored. Neither 231-BR or 231 increase migration towards a Dkk-1 gradient. Instead, both increase migration when directly stimulated with Dkk-1 and then exposed to an external chemotactic gradient of FBS, indicating cancer exposure to Dkk-1 produced within the BBN may promote cancer cell migration. Strikingly, Dkk-1 neutralization in BBN chips with astrocytes reduces 231-BR extravasation across the BBB, decreases migration within the brain niche space, and disrupts cancer–astrocyte contacts, suggesting Dkk-1 is a critical cytokine that promotes brain metastasis. Thirty genes were significantly differentially expressed with Dkk-1 stimulation. The top 3, MDA-231-BR expressed genes FGF-13, PLCB1, and MYC are involved in important oncological pathways: Ras, PI3K, MAPK, Wnt suggesting the plasticity of metastatic cancer permits the cells to adapt to the brain TME. An FGF-13 knockdown of the cells showed Dkk1 effects are partially reduced migration in the brain tropic cells in Dkk1 stimulated conditions. We conclude that the cancer response to Dkk-1 is one critical interaction amenable to therapeutics. Citation Format: Christopher Ryan Oliver, Trisha M. Westerhof, Benjamin A. Yang, Nathan M. Merrill, Joel A. Yates, Liam Russell, Anna J. Miller, Peter J. Ulintz, Carlos A. Aguilar, Aki Morikawa, Maria G. Castro, Sofia M. Merajver. Characterization of secretory cues that promote brain metastasis using a microfluidic blood brain niche (BBN) device [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 3189.

Abstract 3223: Prospective study for the assessment of exosomes in ascitic fluid as prognostic markers in patients with advanced ovarian cancer

Abstract Background: Exosomes have been involved in tumor progression and the development of therapeutic resistances in several malignancies. These extracellular vesicles (EVs) promote angiogenesis, premetastatic niches formation and the communication between the tumor and its environment. Ovarian cancer (OC) is a highly lethal tumor that typically spreads within the abdominal cavity rather than through the blood vessels. Based on this behavior, we aimed to study the presence of exosomes in ascitic fluid of OC patients and explore their potential as predictors of tumor volume and chemoresistance. Methods: We designed an observational prospective study in advanced OC patients who underwent a diagnostic laparoscopy or cytoreductive surgery at our institution. Abdominal washings of a cohort of non-cancer patients were collected as controls. All cases provided written consent. Clinical data and tumor genomic alterations (NGS, FoundationOne CDx) were recorded. EVs pellets were collected by ultracentrifugation and profiled by NTA and western blot (WB) (EVs markers: CD9, TSG101 and ALIX; non EVs related markers: ALB and APOB). PAX8, a protein characteristically expressed in OC cells, was used as a marker of tumor EVs. EVs proteins were quantified by BCA assay; mass spectrometry profiling is ongoing. Results: 45 peritoneal fluids from 40 OC patients (median age [range] 62 [44-79] years) and 29 peritoneal washings from controls have been collected. Our cohort comprise various histological subtypes (high-grade serous, n=32 [80%]; low-grade serous, n=2 [5%]; endometrioid, n=3 [8%)]; clear cells, n=2 [5%] and mucinous, n=1 [2%]) and tumor stages (TI-II, n=5 [12%]; TIII; n=24 [60%]; TIV, n=11 [28%]). Samples were collected either from primary, n=18 (40%); interval, n=15 (33%); or relapse surgery, n=12 (27%). Pathogenic BRCA events were detected in 25% of patients. Mean concentration of exosomes (MCE) was 6.5x106 vs. 3.2x106 particles/ml (p/ml) in cases vs. controls (p<0.0005). No differences in MCE were observed regarding stages I-II vs. stages III-IV (7.1x106 vs. 6.5x106 p/ml; p=0.84), BRCA status (mutant=6.9x106 vs. wild type=6.8x106 p/ml, p=0.46) or platinum response (resistant=7.7x106 vs. sensitive=9.5x106 p/ml, p=0.14). WB analysis showed an enrichment in EVs markers from cancer patients vs. controls (>3-fold). Protein mean concentration in EVs of cancer patients vs. controls was 0.85µg/µl and 0.51µg/µl respectively (p=0.04). Proteomic characterization through mass spectrometry is ongoing and will be presented at the time of the meeting. Conclusions: Our data, although preliminary, show a higher concentration of EVs and EVs proteins in OC cases vs controls with no difference between stages or genomic backgrounds. These results lead to the notion that exosomes could be involved in OC evolution and spread regardless of tumor volume or the molecular subtype. Citation Format: Miguel Quiralte, Arantzazu Barquin, Elena Sevillano, Paloma Navarro, Monica Yagüe, Maria Barba, Juan F. Rodriguez-Moreno, Alejandra Balarezo, Héctor Peinado, Elena Izquierdo, Jesús García-Donas, Sergio Ruiz-Llorente. Prospective study for the assessment of exosomes in ascitic fluid as prognostic markers in patients with advanced ovarian cancer [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 3223.

Abstract 3138: The lung pro-thrombotic niche drives cancer-associated thromboembolism via exosomal ITGB2

Abstract Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in lung cancer and pancreatic ductal adenocarcinoma (PDAC), and in advanced-stage and metastatic cancers. Despite the benefits associated with thromboprophylaxis for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Exosomes are small circulating extracellular vesicles that mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of exosomes into the blood circulation and have displayed a therapeutic and predictive value in systemic diseases. Integrins expressed on the surface of exosomes drive their selective organotropism and prepare distant sites for metastatic seeding by establishing favorable pre-metastatic niches. Here we show that exosomes from metastasis-bearing lungs or pre-metastatic lungs of mice with melanoma, breast, lung and pancreatic cancer induce TE in mice and express high levels of integrin beta 2 (ITGB2). Instead, exosomes from tumor cell lines, primary tumors or other metastasis-bearing organs did not show any pro-thrombotic properties. Myeloid cells including monocytes/macrophages and neutrophils infiltrating pre- and post-metastatic lungs were the main source of ITGB2+ pro-thrombotic exosomes. Blockade of ITGB2 on lung-derived exosomes, or systemically in mice, prevented exosome-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that exosomal ITGB2 interact directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that exosomal ITGB2 levels are elevated in the plasma of PDAC patients prior to TE events in comparison to PDAC patients with no history of TE, and thus might serve as prognostic biomarker of TE. Together, our results provide the first evidence of the establishment of a pro-thrombotic lung niche in different cancer types. Moreover, we identify exosomal ITGB2 as a new target for the prevention and/or treatment of TE, as well as a potential “liquid biopsy” analyte for the early stratification of patients at high risk of TE. Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soren Heissel, Harry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David R. Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism via exosomal ITGB2 [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 3138.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Evaluation of miRNA-21-5p and miRNA-10b-5p levels in serum-derived exosomes of breast cancer patients in different grades

Background & objectivesTumor cells have various effects and dominance over other healthy cells. Cancer cells alter the cell program in healthy cells by secreting exosomes containing microRNAs involved in epithelial-mesenchymal transition (EMT). They can migrate to distant organs and establish a pre-metastatic niche. The purpose of this study was to determine the expression of miRNA-21–5p and miRNA-10b-5p, both of which are involved in EMT, in breast cancer-derived exosomes of various grades in order to identify new biomarkers involved in breast cancer progression. MethodsIn this study, a blood sample was taken from 60 patients with grades I, II, or III breast cancer, as well as twenty healthy individuals as a control group. The exosomes were then purified from serum samples, and their relative expression of miRNA-21–5p and miRNA-10b-5p was determined using the real-time PCR method. ResultsmiRNA-21–5p expression was significantly increased in patients with breast cancer grades I, II, and III compared to the control group (p < 0.01), (p < 0.0001) and (p < 0.0001), respectively, as was miRNA-10b-5p expression in patients with breast cancer grades I, II, and III compared to the control group (p < 0.0001), (p < 0.0001) and (p < 0.0001), respectively. ConclusionOur results show that both microRNAs increase as cells lose their differentiation and become more invasive, which is evidence of cancer progression. Hence, both microRNAs may have the potential to be used alone or in combination with other biomarkers for the diagnosis and prognosis of breast cancer.

Peptide nano-blanket impedes fibroblasts activation and subsequent formation of pre-metastatic niche

There is evidence to suggest that the primary tumor induces the formation of a pre-metastatic niche in distal organs by stimulating the production of pro-metastatic factors. Given the fundamental role of the pre-metastatic niche in the development of metastases, interruption of its formation would be a promising strategy to take early action against tumor metastasis. Here we report an enzyme-activated assembled peptide FR17 that can serve as a “flame-retarding blanket” in the pre-metastatic niche specifically to extinguish the “fire” of tumor-supportive microenvironment adaption. We show that the in-situ assembled peptide nano-blanket inhibits fibroblasts activation, suppressing the remodeling of the metastasis-supportive host stromal tissue, and reversing vascular destabilization and angiogenesis. Furthermore, we demonstrate that the nano-blanket prevents the recruitment of myeloid cells to the pre-metastatic niche, regulating the immune-suppressive microenvironment. We show that FR17 administration effectively inhibits the formation of the pulmonary pre-metastatic niche and postoperative metastasis, offering a therapeutic strategy against pre-metastatic niche formation.

Revisiting laminin and extracellular matrix remodeling in metastatic squamous cell carcinoma: What have we learned after more than four decades of research?

Patients with squamous cell carcinoma (SCC) have significantly lower survival upon the development of distant metastases. The extracellular matrix (ECM) is a consistent yet dynamic influence on the metastatic capacity of SCCs. The ECM encompasses a milieu of structural proteins, signaling molecules, and enzymes. Just over 40 years ago, the fibrous ECM glycoprotein laminin was identified. Roughly four decades of research have revealed a pivotal role of laminins in metastasis. However, trends in ECM alterations in some cancers have been applied broadly to all metastatic diseases, despite evidence that these characteristics vary by tumor type. We will summarize how laminins influence the SCC metastatic process exclusively. Enhanced laminin protein deposition occurs at the invasive edge of SCC tumors, which correlates with elevated levels of laminin-binding β1 integrins on SCC cells, increased MMP-3 presence, worse prognosis, and lymphatic dissemination. Although these findings are significant, gaps in knowledge of the formation of a premetastatic niche, the processes of intra- and extravasation, and the contributions of the ECM to SCC metastatic cell dormancy persist. Bridging these gaps requires novel in vitro systems and animal models that reproduce tumor-stromal interactions and spontaneous metastasis seen in the clinic. These advances will allow accurate assessment of laminins to predict responders to transforming growth factor-β inhibitors and immunotherapy, as well as potential combinatorial therapies with the standard of care. Such clinical interventions may drastically improve quality of life and patient survival by explicitly targeting SCC metastasis.

Receptor Activity Modifying Protein RAMP Sub-Isoforms and Their Functional Differentiation, Which Regulates Functional Diversity of Adrenomedullin.

Simple SummaryThe body contains a variety of humoral bioactive factors and information regulation systems that maintain homeostasis. Although adrenomedullin (AM) was first identified as a vasodilator peptide, subsequent studies have revealed that it plays an essential role in homeostasis in vivo and possesses a variety of bioactivities other than vasodilation. Based on its structural homology, AM is classified as a member of the calcitonin superfamily, which includes calcitonin, calcitonin gene-related peptide (CGRP), amylin, and intermedin. Peptides belonging to the calcitonin superfamily are characterized by a partial sharing of their receptor system. One of the sub-isoforms of receptor activity-modifying protein (RAMP) binds to calcitonin receptor-like receptor (CLR), a 7-transmembrane G protein, in a one-to-one manner, and then functions as a receptor for these peptides. Among RAMP isoforms, we focused on RAMP2 and RAMP3. Each RAMP isoform is expected to be a potential therapeutic target for various diseases, including cardiovascular diseases and cancer metastasis. In this review, we outline the pathophysiological significance and functional differentiation of RAMP2 and RAMP3, as mainly revealed by the analysis of genetically engineered mice.AM knockout (AM-/-) and RAMP2 knockout (RAMP2-/-) proved lethal for mice due to impaired embryonic vascular development. Although most vascular endothelial cell-specific RAMP2 knockout (E-RAMP2-/-) mice also died during the perinatal period, a few E-RAMP2-/- mice reached adulthood. Adult E-RAMP2-/- mice developed spontaneous organ damage associated with vascular injury. In contrast, adult RAMP3 knockout (RAMP3-/-) mice showed exacerbated postoperative lymphedema with abnormal lymphatic drainage. Thus, RAMP2 is essential for vascular development and homeostasis and RAMP3 is essential for lymphatic vessel function. Cardiac myocyte-specific RAMP2 knockout mice showed early onset of heart failure as well as abnormal mitochondrial morphology and function, whereas RAMP3-/- mice exhibited abnormal cardiac lymphatics and a delayed onset of heart failure. Thus, RAMP2 is essential for maintaining cardiac mitochondrial function, while RAMP3 is essential for cardiac lymphangiogenesis. Transplantation of cancer cells into drug-inducible vascular endothelial cell-specific RAMP2 knockout mice resulted in enhanced metastasis to distant organs, whereas metastasis was suppressed in RAMP3-/- mice. RAMP2 suppresses cancer metastasis by maintaining vascular homeostasis and inhibiting vascular inflammation and pre-metastatic niche formation, while RAMP3 promotes cancer metastasis via malignant transformation of cancer-associated fibroblasts. Focusing on the diverse physiological functions of AM and the functional differentiation of RAMP2 and RAMP3 may lead to the development of novel therapeutic strategies.

MiR-26b-5p in small extracellular vesicles derived from dying tumor cells after irradiation enhances the metastasis promoting microenvironment in esophageal squamous cell carcinoma

Radiation therapy is effective in achieving local control in esophageal squamous cell carcinoma; however, changes in the tumor microenvironment induced by radiation can also promote metastasis. Dying tumor cells play vital roles in promoting the survival of living tumor cells; however, few studies have investigated the effects of dying tumor cells on the tumor microenvironment. Since myeloid-derived suppressor cells (MDSCs) and macrophages constitute the pre-metastatic niche (PMN), we used a 4-nitroquinoline-1-oxide induced in situ tumor model to investigate the effects of irradiation on MDSCs and macrophages in esophageal squamous cell carcinoma (ESCC). When primary tumor sites were irradiated, we observed an increase in MDSCs in the spleen and the deposition of PMN components in lung and liver. Enhanced MDSC accumulation and function were induced by small extracellular vesicles (sEVs) isolated from irradiated tumor-bearing mice. The MDSC induction function of sEVs after irradiation was reaffirmed using sEVs derived from ESCC cell lines. The irradiation-induced upregulation of miR-26b-5p in sEVs enhanced MDSC expansion and activation by targeting phosphatase and tensin homolog. Our results first elucidated a mechanism by which dying tumor cells enhanced the deposition of PMN components and potentiated MDSCs in ESCC after irradiation. sEVs played a vital role in mediating signals between the primary tumor and the microenvironment to form a metastasis-promoting microenvironment after irradiation. Furthermore, miR-26b-5p or PI3K/AKT signaling pathway inhibitors should be evaluated in clinical trials in combination with radiotherapy as a strategy to improve outcomes.

Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog “in cascade”, between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

From Tumor Cells to Endothelium and Gut Microbiome: A Complex Interaction Favoring the Metastasis Cascade.

Metastasis is a complicated process through which tumor cells disseminate to distant organs and adapt to novel tumor microenvironments. This multi-step cascade relies on the accumulation of genetic and epigenetic alterations within the tumor cells as well as the surrounding non-tumor stromal cells. Endothelial cells constitute a major player in promoting metastasis formation either by inducing the growth of tumor cells or by directing them towards dissemination in the blood or lymph. In fact, the direct and indirect interactions between tumor and endothelial cells were shown to activate several mechanisms allowing cancer cells’ invasion and extravasation. On the other side, gastrointestinal cancer development was shown to be associated with the disruption of the gut microbiome. While several proposed mechanisms have been investigated in this regard, gut and tumor-associated microbiota were shown to impact the gut endothelial barrier, increasing the dissemination of bacteria through the systemic circulation. This bacterial dislocation allows the formation of an inflammatory premetastatic niche in the distant organs promoting the metastatic cascade of primary tumors. In this review, we discuss the role of the endothelial cells in the metastatic cascade of tumors. We will focus on the role of the gut vascular barrier in the regulation metastasis. We will also discuss the interaction between this vascular barrier and the gut microbiota enhancing the process of metastasis. In addition, we will try to elucidate the different mechanisms through which this bacterial dislocation prepares the favorable metastatic niche at distant organs allowing the dissemination and successful deposition of tumor cells in the new microenvironments. Finally, and given the promising results of the studies combining immune checkpoint inhibitors with either microbiota alterations or anti-angiogenic therapy in many types of cancer, we will elaborate in this review the complex interaction between these 3 factors and their possible therapeutic combination to optimize response to treatment.

CD73/Adenosine Pathway Involvement in the Interaction of Non-Small Cell Lung Cancer Stem Cells and Bone Cells in the Pre-Metastatic Niche.

Adenosinergic signaling is an important regulator of tissue homeostasis and extracellular accumulation of adenosine (Ado) and is associated with different pathologies, such as cancer. In non-small-cell lung cancer (NSCLC), a subset of CD133/CXCR4+ cancer stem cell (CSCs) has been demonstrated to initiate bone metastases. Here we investigated how NSCLC CSCs interact with osteoclasts (OCs) and osteoblasts (OBs) by modulating Ado production and OC activity. We proved that CSC-spheres, generated in vitro from NSCLC cell lines, express CD38, PC-1, and CD73, enzymes of the non-canonical adenosinergic pathway, produce high level of Ado, and down-regulate A1R and A3R inhibitory receptors, while expressing A2AR and A2BR. To address the Ado role and modulation of the in-bone pre-metastatic niche, we performed co-cultures of CSC-spheres with OCs and OBs cells. Firstly, we verified that active OCs do not activate non-canonical the adenosinergic pathway, conversely to OBs. OCs co-cultured with CSC-spheres increase Ado production that is related to the OC resorption activity and contributes to T-cell suppression. Finally, we proved the efficacy of anti-CD73 agents in blocking NSCLC cell migration. Overall, we assessed the importance of adenosinergic signaling in the interaction between CSCs and OCs at the pre-metastatic niche, with therapeutic implications related to Ado production.

Phenotypic and transcriptomic profiles of tumor-conditioned macrophages in dogs, and the translational value of canine cancer immunology research.

Abstract Tumor-associated macrophages (TAMs) are heterogeneous and abundantly present in the tumor stroma. They derive from tissue-resident macrophages and recruited monocytes and play a central role in several aspects of cancer. In murine models, TAMs promote invasion, intravasation, circulatory survival and extravasation of tumor cells. TAMs also promote tumor progression and angiogenesis, remodel the tumor microenvironment, and modulate the adaptive immune system. Evidence suggests TAMs are vital for establishing the premetastatic niche. Historically, family-owned dogs have proven to be reliable cancer models. In contrast to laboratory animals, dogs develop cancer naturally and commonly, co-inhabit our environment, are genetically outbred and immunologically experienced. We hypothesized that canine tumor conditioned monocyte-derived macrophages (TCMs) share many characteristics with M2 macrophages and have a tumor-promoting phenotype as in humans. We collected blood from healthy dogs, isolated peripheral blood monocytes and differentiated them in vitro using tumor conditioned media from three canine cancer lines and M-CSF. Three TCM populations were generated and compared with M1 and M2 macrophages. We assessed the macrophages using multicolor flow cytometry and RNA-sequencing. All TCM showed an increased expression of two M2 markers (CD209a and FcɛRI), while 2/3 TCM populations had increased expression of two additional M2 markers (CD206 and CD11d). Transcriptomic analysis is ongoing. Results so far support the hypothesis that canine TCMs share many similarities with human TCMs, further warranting the use of dogs as cancer immunology models. Supported by grant from Agria Animal Insurances (N-2020-0061) and The Research fund for cancer in dogs (Oslo, Norway)

Immunosuppressive Extracellular Vesicles as a Linking Factor in the Development of Tumor and Endometriotic Lesions in the Gynecologic Tract.

Both gynecological tumors and endometriosis require for their development a favorable environment, termed in the case of tumors a “pre-metastatic niche” and in case of endometriosis a “pro-endometriotic niche”. This is characterized by chronic inflammation and immunosuppression that support the further progression of initial lesions. This microenvironment is established and shaped in the course of a vivid cross-talk between the tumor or endometrial cells with other stromal, endothelial and immune cells. There is emerging evidence that extracellular vesicles (EVs) play a key role in this cellular communication, mediating both in tumors and endometriosis similar immunosuppressive and pro-inflammatory mechanisms. In this review, we discuss the latest findings about EVs as immunosuppressive factors, highlighting the parallels between gynecological tumors and endometriosis. Furthermore, we outline their role as potential diagnostic or prognostic biomarkers as well as their future in therapeutic applications.