Tumor-associated Stromal Cells Research Articles

Latest changes in the molecular classification of breast cancer and triple negative breast cancer

Breast cancer (BC) is a heterogeneous disease including multiple histological subtypes with different biological behavior and clinical outcome for the patient. Due to its heterogeneity, BC is classified into different groups, providing possibilities for target therapies and improving the outcome – histological, TNM classification, molecular classification. The molecular classification, proposed at the beginning by Perou et al in 2000, divides BC into 5 subtypes - Luminal A, Luminal B, HER 2 enriched, claudin-low and basal like. More attention should be paid to the further classification of basal like BC. This is also a heterogeneous disease including triple negative breast cancers (TNBC). They show no expression of ER, PrR or HER2 and are considered difficult to treat. Despite the bad prognosis, some of these patients demonstrate great benefit from the traditional neoadjuvant chemotherapy. Lehman et al. identified 6 molecular subtypes of TNBC, using gene expression profiling – basal like 1 (BL1), basal like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM) and luminal androgen receptor (LAR). However, after histopathological quantification and laser-capture microdissection they refined their classification and in 2016 the new molecular classification of TNBC excluding the IM and MSL types was published. The working group has concluded that the characteristics of the tumors in these two groups were due to tumor-infiltrating lymphocytes and tumor-associated stromal cells and not to the tumor cells. The refinement of the classification of TNBC provides a better distribution of the patients into the different groups and better response to the applied therapy.

The Interplay between Colon Cancer Cells and Tumour-Associated Stromal Cells Impacts the Biological Clock and Enhances Malignant Phenotypes.

Cancer cells interrelate with the bordering host microenvironment that encompasses the extracellular matrix and a nontumour cellular component comprising fibroblasts and immune-competent cells. The tumour microenvironment modulates cancer onset and progression, but the molecular factors managing this interaction are not fully understood. Malignant transformation of a benign tumour is among the first crucial events in colorectal carcinogenesis. The role of tumour stroma fibroblasts is well-described in cancer, but less well-characterized in benign tumours. In the current work we utilized fibroblasts isolated from tubulovillous adenoma, which has high risk for malignant transformation, to study the interaction between benign tumour stroma and the circadian clock machinery. We explored the role of the biological clock in this interplay taking advantage of an experimental model, represented by the co-culture of colon cancer cells with normal fibroblasts or tumour-associated fibroblasts, isolated from human colorectal tumour specimens. When co-cultured with tumour-associated fibroblasts, colon cancer cells showed alterations in their circadian and metabolic parameters, with decreased apoptosis, increased colon cancer cell viability, and increased resistance to chemotherapeutic agents. In conclusion, the interactions among colon cancer cells and tumour-associated fibroblasts affect the molecular clockwork and seem to aggravate malignant cell phenotypes, suggesting a detrimental effect of this interplay on cancer dynamics.

Abstract 4965: Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer

Abstract Immunotherapy has proven to be a powerful anti-tumor therapy, harnessing the body’s own immune system to target and kill tumor cells. However, immunotherapy is not successful in all cancer patients due to both intrinsic non-responsiveness and adaptive resistance. Developing predictive biomarkers and understanding mechanisms of resistance are major goals of the immuno-oncology community. The extracellular matrix (ECM), an important factor for promoting tumor growth, survival, and migration of tumor cells, can also act as a physical barrier to prevent immune cell infiltration and promote tumor immune escape. Components of the ECM such as COL11A1, COL4A1, and LOXL2 have been shown to be associated with cancer progression. Furthermore, new data suggests that TGFβ activation leads to up-regulation of ECM genes in cancer-associated fibroblasts and immune suppression. However, it remains poorly understood which cells in the tumor microenvironment (TME) are the sources of ECM gene expression and how they are related to tumor infiltrating cytotoxic T lymphocytes (CTLs). In this study, we employed a highly sensitive and specific RNAscope in situ hybridization (ISH) duplex assay to directly visualize the tissue distribution of cells expressing COL4A1, COL11A1, LOXL2, and TGFB1 in relation to tumor infiltrating CTLs in non-small cell lung carcinoma (NSCLC). NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE tumor samples were analyzed using this ISH assay with the following probe combinations: Hs-CD8/Hs-IFNG, Hs-CD4/Hs-FOXP3, Hs-LOXL2/Hs-COL4A1, and Hs-TGFB1/Hs-COL11A1. We observed COL4A1 expression in both tumor and tumor-associated stromal cells in different samples. In contrast, COL11A1 was only expressed in tumor-associated stromal cells. Interestingly, high COL4A1 expression was associated with high CD8+ T cell infiltration, whereas high COL11A1 expression was associated with poor CD8+ T cell infiltration. In addition, tumor expression of TGFB1 was positively correlated with COL11A1 expression. These data depict a complex landscape of ECM gene expression and their relationship to T cell infiltration in the tumor and TME. Taken together, these results demonstrate that the RNAscope assay provides a powerful approach to directly examine the interactions between tumor, ECM, and T cell immune infiltration, and offers advantages over immunohistochemistry (IHC) for identifying the cellular sources of secreted proteins such as ECM components in the TME. Citation Format: NA LI, Hongzhe Sun, Xin Wang, Zhifu Zhang, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4965.

Abstract 3890: Pharmacodynamic and proteomic analysis of combined gemcitabine/nab-paclitaxel in patient-derived pancreatic cancer xenograft models

Abstract Gemcitabine (GEM) combined with albumin-bound paclitaxel nanoparticles (nab-PTX) is the first-line therapy for patients with metastatic pancreatic adenocarcinoma (PDAC). However, the in vivo mechanisms of drug action and interaction have not been investigated quantitatively for this combination. We evaluated the effects of GEM (100 mg/kg iv weekly) and nab-PTX (equivalent to 30 mg/kg PTX iv weekly), alone or combined, on tumor volume progression in 3 patient-derived xenograft (PDX) PDAC models in SCID mice. The MS1-based quantitative proteomic analysis (IonStar), which offers highly reproducible and accurate quantification with extremely low missing data (<0.5%), was conducted on Days 1, 4, and 8 after dosing to evaluate tumor pharmacodynamic responses in protein expression. Proteins were quantified based on species-specific peptides in order to identify responses of the PDAC cells (human) and tumor-associated stromal cells (murine). A population pharmacokinetic/pharmacodynamic model was developed to quantify tumor growth inhibition by the individual and combined drugs. Tumor growth kinetics were described by a composite function of exponential growth transitioning to linear growth. Treatment effects were represented by a 2nd-order, concentration-dependent parameter k that leads to cell death following delays arising from signal transduction. All 3 PDX models responded to the combination therapy, but with different sensitivities. PDX#14312 showed the least inhibition by the drugs alone (k[GEM]=13.6, k[PTX]=37.0 /day/mM), compared to the other PDXs (#18254: k[GEM]=68.1, k[PTX]=125; 18269: k[GEM]=112, k[PTX]=51.2 /day/mM). Compared to the more potent single-drug treatment for each PDX, the combination resulted in 1.33-fold greater cell death in #14312, 1.39 fold in #18269 and 1.19 fold in #18254. Model simulations showed distinct temporal profiles of cytotoxic signals in the 3 PDXs. Pathway analysis showed that the top changes mediated by the drug combination in PDX#14312 PDAC cells included extracellular matrix organization (FDR-corrected p-value = 7E-5), synthesis of nucleotide di- and triphosphates (p = 0.012), and interferon signaling (p = 0.04), whereas top changes in stroma included increased platelet activation (p = 3.8E-4) and extracellular matrix organization (p = 0.046). These processes represent potential contributors to the resistance of PDX#14312 to the drug combination. Top changes in #18269 and #18254, which were sensitive to the combination, included increased glycolysis (p = 0.038), decreased fatty acid beta-oxidation (p = 3E-5) and DNA replication (p = 0.0058) in PDAC cells, and increased complement activation (p < 1E-5) in stroma. Overall, inter-patient variability in PDAC responsiveness to GEM/nab-PTX therapy may be associated with tumor stress responses partially resembling wound healing processes. Citation Format: Jin Niu, Xue Wang, Shichen Shen, Ju Qu, Donald Mager, Robert M. Straubinger. Pharmacodynamic and proteomic analysis of combined gemcitabine/nab-paclitaxel in patient-derived pancreatic cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3890.

Abstract 2821: Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer

Abstract Tumor infiltration by immune cells critically impacts on clinical outcome of human colorectal cancer (CRC). While high densities of CD8+ T cells and T-helper type 1 cells are associated with prolonged patient survival, the role of tumor-associated neutrophils (TANs) is debated. CRC arise in an environment heavily populated by microrganisms. Upon CRC oncogenesis, gut commensal bacteria translocate into the submucosa where they directly interact with residing immune cells. Neutrophils represent a front-line arm of the immune system in the response to bacteria. However, little is known about their interaction with commensal bacteria within CRC tissues. We investigated the interplay between neutrophils and commensal bacterial species present within the CRC microenvironment. Using an orthotopic CRC xenograft model, we found that commensal bacteria stimulate CRC cells to produce neutrophil recruiting chemokines. We then compared in vitro chemokine induction capacity of Fusobacterium nucleatum (Fn) and Bacteroides fragilis (Bf), two most abundant bacterial species of CRC microenvironment. Fninduced significantly higher levels of IL-8 by CRC cells than Bf, thus more effectively promoting neutrophil recruitment. Accordingly, in human CRC samples, abundance of Fn, but not Bf, significantly correlated with high infiltration by CD66b+ cells. Functional studies indicate that neutrophils cultured in the presence of Fn, but not Bf, lose their ability to enhance proliferation and cytokine release by CD8+ T cells undergoing antigenic stimulation. Moreover, neutrophils exposed to Fn, but not Bf, stimulate release of IL-6 by tumor-associated stromal cells, leading to enhanced tumor cell proliferation. Our data cumulatively suggest that distinct bacterial components of the human gut flora might differentially modulate functions of CRC infiltrating neutrophils, thus ultimately influencing their prognostic significance. Note: This abstract was not presented at the meeting. Citation Format: Valeria Governa, Eleonora Cremonesi, Diego Calabrese, Valentina Mele, Emanuele Trella, Raoul A. Droeser, Martin Bolli, Serenella Eppenberger-Castori, Salvatore Piscuoglio, Charlotte K. y. Ng, Jesus Francisco Glaus Garzon, Lubor Borsig, Klaus-Peter Janssen, Luigi M. Terracciano, Giulio Cesare Spagnoli, Dimitri Christoforidis, Pietro Edoardo Majno-Hurst, Giandomenica Iezzi. Gut commensal bacteria modulate functions of tumor-associated neutrophils in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2821.

Interleukin‐8 is Released from Human Pancreatic Tumor and Stromal Cells, and Causative in Skeletal Muscle Atrophy

Cancer cachexia is a debilitating metabolic disorder characterized by loss of body weight, muscle mass, and functional deterioration that negatively affects patient's quality of life. Cachexia is particularly prevalent in patients with pancreatic cancer; present in more than 60% of patients at time of diagnosis and more than 80% of advanced stage patients. Initiating factors of cancer cachexia are tumor‐derived, at least in part, yet our understanding of the specific factors is still far from complete. Given that cancer cells collaborate in the tumor microenvironment with tumor associated stromal cells, we collected conditioned media (CM) from primary human pancreatic cancer (PhPC) cells, primary pancreatic tumor associated stromal (PPTAS) cells, and their co‐culture and measured selected cytokines and chemokines using multiple analyte profiling. Amongst all factors measured in PhPC/PPTAS co‐culture CM, Interleukin‐8 (IL‐8/CXCL8) was the highest at 6,071 pg/ml. IL‐8 levels in PhPC CM and PPTAS CM were 625 pg/ml and 71 pg/m, respectively. Thus, there is a synergistic increase in IL‐8 secretion when human pancreatic cancer and stromal cells communicate physically and/or biochemically. To determine the extent to which IL‐8 may cause muscle atrophy we treated differentiated C2C12 myotubes or human myotubes with recombinant IL‐8 (rIL‐8) for 48 hours, which caused a 28% and 25% reduction in myotube diameter, respectively. To test whether PhPC cell CM induces myotube atrophy via IL‐8 signaling we treated C2C12 myotubes with PhPC CM, with or without IL‐8 neutralization or inhibition of the IL‐8 receptor, CXCR2. Myotube diameter was significantly decreased 29% in PhPC CM treated myotubes, but this was significantly attenuated with IL‐8 neutralization and completely abolished by CXCR2 inhibition. To determine whether IL‐8 can lead to muscle wasting in vivo, we injected 50mg/kg of rIL‐8 i.p. into mice every other day for 6 days. This increased serum IL‐8 levels and caused a reduction in body weight (−11%), tibialis anterior (TA) weight (−17%), tricep surae weight (−15%), and gonadal fat weight (−34%). Furthermore, the mean muscle fiber cross sectional area of the TA was also significantly decreased and microarray analysis on these muscles identified ubiquitin ligase complex and oxidative phosphorylation as two of the most enriched biological processes among upregulated and downregulated genes, respectively. These findings identify IL‐8 as a chemokine released from human pancreatic cancer and stromal cells that is sufficient to cause myotube atrophy, and as an active factor causative in myotube atrophy by PhPC CM that is sufficient to induce a cachexia phenotype in vivo.Support or Funding InformationThis work was supported by a Florida Department of Health, Biomedical Research Program Grant, 7BC02 (to A.R.J). C.C.C. and R.L.N. are supported by NIH, NICHD, T32HD043730.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Seneca Valley Virus Exploits TEM8, a Collagen Receptor Implicated in Tumor Growth.

Recent studies reveal that Seneca Valley Virus (SVV) exploits tumor endothelial marker 8 (TEM8) for cellular entry, the same surface receptor pirated by bacterial-derived anthrax toxin. This observation is particularly significant as SVV is a known oncolytic virus which selectively infects and kills tumor cells, particularly those of neuroendocrine origin. TEM8 is a transmembrane glycoprotein that is preferentially upregulated in some tumor cell and tumor-associated stromal cell populations. Both TEM8 and SVV have been evaluated for targeting of tumors of multiple origins, but the connection between the two was previously unknown. Here, we review currently understood interactions between TEM8 and SVV, anthrax protective antigen (PA), and collagen VI, a native binding partner of TEM8, with an emphasis on potential therapeutic directions moving forward.

Stromal Cell PD-L1 Inhibits CD8+ T-cell Antitumor Immune Responses and Promotes Colon Cancer.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFα-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8+ granzyme B-secreting T cells in vitro, and the inhibition of CD8+ effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome from human colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8+ antitumor immune responses and potentiation of colorectal cancer progression. Cancer Immunol Res; 6(11); 1426-41. ©2018 AACR.

Abstract 2130: Novel ADAM9 variant induces prostate cancer metastasis

Abstract Prostate cancer often progresses from an androgen to castration-refractory prostate cancer and metastasizes to the bone and lymph nodes. However, there is no biomarker to predict the initiation of cancer metastasis. Therefore, it is an unmet need to understand molecules which induce metastasis to replace the prediction by PSA. Previous studies by us demonstrate the elevation of ADAM9 in PCa malignant progression. However, the role of ADAM9 in the tumor microenvironment is still not fully understood. Using laser capture microdissection of prostate tumor and stromal cells, indicating the increased of ADAM9 and reactive oxygen species in tumor-associated stromal cells. These inductions correlated with the increased expression of SOD-2 in tumor-associated stromal cells. Both electron microscope analysis and conditioned medium of stromal cells demonstrated the increased of soluble ADAM9 level in the medium. ELISA analysis of patient serum confirmed the increased of ADAM9 serum levels in prostate cancer patients. PCR and sequencing analysis confirmed a novel soluble ADAM9 (sADAM9v2) with deletion of membrane domain that induces cell migration through the activation of FAK and AKT phosphorylation. Our studies demonstrated the release of sADAM9 in tumor microenvironment correlated with reactive oxygen stress and regulate prostate cancer migration and metastasis. In conclusion, our studies showed that sADAM9 release by cancer-associated stromal cells could be a potential biomarker for prostate cancer progression. Citation Format: Chia-Ling Hsieh, Shian-Ying Sung. Novel ADAM9 variant induces prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2130.

Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.

Stromal cells in breast cancer as a potential therapeutic target.

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

Su1319 - Culture Model Development for Pancreatic Cancer Stem Cells and Tumor-Associated Stromal Cells from EUS-Guided Fine Needle Aspirates

Su1319 - Culture Model Development for Pancreatic Cancer Stem Cells and Tumor-Associated Stromal Cells from EUS-Guided Fine Needle Aspirates

Contribution to Tumor Angiogenesis From Innate Immune Cells Within the Tumor Microenvironment: Implications for Immunotherapy.

The critical role of angiogenesis in promoting tumor growth and metastasis is strongly established. However, tumors show considerable variation in angiogenic characteristics and in their sensitivity to antiangiogenic therapy. Tumor angiogenesis involves not only cancer cells but also various tumor-associated leukocytes (TALs) and stromal cells. TALs produce chemokines, cytokines, proteases, structural proteins, and microvescicles. Vascular endothelial growth factor (VEGF) and inflammatory chemokines are not only major proangiogenic factors but are also immune modulators, which increase angiogenesis and lead to immune suppression. In our review, we discuss the regulation of angiogenesis by innate immune cells in the tumor microenvironment, specific features, and roles of major players: macrophages, neutrophils, myeloid-derived suppressor and dendritic cells, mast cells, γδT cells, innate lymphoid cells, and natural killer cells. Anti-VEGF or anti-inflammatory drugs could balance an immunosuppressive microenvironment to an immune permissive one. Anti-VEGF as well as anti-inflammatory drugs could therefore represent partners for combinations with immune checkpoint inhibitors, enhancing the effects of immune therapy.

CXCL14 and NOS1 expression in specimens from patients with stage I-IIIA nonsmall cell lung cancer after curative resection.

Many studies show that CXC chemokine ligand 14 (CXCL14) is highly expressed in tumor-associated stromal cells, promoting tumor cell growth, and invasion. Because of its unclear receptors, CXCL14-initiated intracellular signal cascades remain largely unknown. However, CXCL14 can regulate nitric oxide synthase 1 (NOS1) as its intracellular molecular target. In this paper, we investigated the expression of CXCL14 and NOS1 in specimens from patients with stage I–IIIA nonsmall cell lung cancer (NSCLC) after curative resection, and evaluated the prognostic significance of this gene expression in stromal fibroblasts and cancer cells.Immunohistochemistry was used to detect the expression of CXCL14 and NOS1 in 106 formalin fixed, paraffin-embedded specimens from patients with stage I-IIIA NSCLC. The chi-square test was performed to examine the correlation of CXCL14 and NOS1 expression level with clinicopathological features. The effects of the expression of CXCL14 or NOS1 on progression-free survival (PFS) and overall survival (OS) were determined by Kaplan–Meier and Cox hazard proportional model.The percentages of high CXCL14 expression in stromal fibroblasts and that in cancer cells were 46.2% (49/106) and 23.6% (25/106), respectively. The positive expression rates of NOS1 in cancer cells were 42.5% (45/106). The result indicated that there was a significant positive correlation between CXCL14 expression level in stromal fibroblasts and that in cancer cells (χ2 = 4.158, P = .041). In addition, the expression of CXCL14 in stromal fibroblasts was significantly correlated with NOS1 expression in cancer cells (χ2 = 16.156, P < .001). The 5-year PFS rates with low and high CXCL14 expression in stromal fibroblasts were 66.7% and 14.3% (χ2 = 44.008, P < .001), respectively, and the 5-year OS rates with those were 87.1% and 43.5% (χ2 = 21.531, P < .001), respectively. The 5-year PFS rates with negative and positive expression of NOS1 in cancer cells were 62.3% and 15.6% (χ2 = 33.756, P < .001), respectively, and the 5-year OS rates with those were 86.4% and 40.1% (χ2 = 24.430, P < 0.01), respectively.Both the high expression of CXCL14 in stromal fibroblasts and the positive expression of NOS1 in cancer cells are independent negative predictors of PFS and OS in patients with stage I–IIIA NSCLC after curative resection.

Biyomoleküllerin Dahi Çocuğu:Eksozomlar ve Akciğer Kanseri Üzerine Bir Bakış

Exosomes; are bioactive receptors which is indirectly originating from the cell membrane. The dimensions vary between 50-140 nm. The most important features distinguishing exosomes from other extracellular vesicles (microvesicles and apoptotic bodies) are their unique biogenesis pathways, lipid compositions, and RNA cargos (mRNA, miRNA, lncRNA) they carry. Exosomes that play a role in cell-cell interaction by virtue of their RNA content can alter the transcriptome and function of the cell with the RNA strands they carry when transferred to the recipient cell. It has also been identified that they carry nucleic acids (DNA, RNA), proteins, nucleoproteins and various enzymes for use in signal transduction. Exosomes are also secreted by cancer cells and tumor-associated stromal cells as they are secreted from healthy cells under physiological conditions. Through the exosomes, autocrine, paracrine and endocrine communication is established between cancer cells. Although exosomal secretion is a normal process, the increase in rate and exosomal mediated transfer of different cargos (oncogenic signals) may mediate oncogenic progression and metastasis. The increase in exosomal quantities and altered cargo expression can be considered as a powerful biomarker for altering normal physiological conditions and can be used to diagnose cancer and many other diseases. Exosomes can be obtained by ultracentrifugation from body fluids such as blood, plasma, cerebrospinal fluid, bile, breast milk, amniotic fluid, saliva, urine and can be evaluated for molecular components such as DNA, RNA, miRNA, and proteins. In addition, as they are derived from the plasma membrane, they are inherently liposomal and nano-sized and can easily move through the blood-brain barrier, due to their protein-lipid content in their membranes. Although exosomes are associated with many types of cancer, they are also important in lung cancer. It has been showed that exosomes are increasing lung endothelial permeability and lung metastasis, and also plays an important role in angiogenesis in lung cancer. As a result, exosomes are smart munchkins carrying cargo between cells and have the ability to convert healthy cells to carcinoma according to the cargo that they carry. They can also be obtained from whole-body fluids and can be used for targeted treatment with their ability to enter the cells easily and to carry them in circulation.

Lipid metabolism and lipophagy in cancer

The tumor microenvironment can be hypoxic, acidic, and deficient in nutrients, thus causing the metabolism of tumor cells as well as the neighboring stromal cells to be remodelled to facilitate tumor survival, proliferation, and metastasis. Abnormal tumor lipid metabolism is a fairly new field, which has received attention in the past few years. Cross-talk between tumor cells and tumor-associated stromal cells modulates the high metabolic needs of the tumor. Fatty acid turnover is high in tumor cells to meet the energy as well as synthetic requirements of the growing tumor. Lipolysis of lipids stored in lipid droplets was earlier considered to be solely carried out by cytosolic lipases. However recent studies demonstrate that lipophagy (autophagic degradation of lipids by acidic lipases) serves as an alternate pathway for the degradation of lipid droplets. Involvement of lipophagy in lipid turnover makes it a crucial player in tumorigenesis and metastasis. In this review we discuss the metabolic reprogramming of tumor cells with special focus on lipid metabolism. We also address the lipid turnover machinery in the tumor cell, especially the lipophagic pathway. Finally, we integrate the current understanding of lipophagy with tumor lipid metabolism.

Caveolin-1, a driver of invasive phenotype in in-vitro 3D-spheroid assays comprised of high grade GBM cells association with an AKT-inhibited phenotype

INTRODUCTION Glioblastoma multiforme (GBM) cells display a highly invasive phenotype, a hallmark which counters effective surgical and radiotherapy strategies. Caveolin-1 (Cav-1) is the main structural and functional component of caveolae. The impact of the expression of Cav-1 within a range of tumour and tumour-associated stromal cells is variable with both oncogenic and tumour suppressive roles reported which appear to be both disease-specific and context-dependent. Our hypothesis is that Cav-1 serves as promoter of invasion of GBM cells. MATHERIALS AND METHODS To investigate our hypothesis we used a lentiviral shRNA approach to silence Cav-1 in three GBM cell lines (U87, UP007, UP029) derived from adult brain tumours. We employed an in-vitro 3D cell-sprouting invasion assay with GBM cell spheres embedded in Matrigel. Quantification of invasion was undertaken using a novel image analysis tool or 3D systems, INSIDIA (ImageJ Macro for High-throughput Spheroid Invasion Analysis). Parallel migration and invasion studies were performed using a Boyden Chamber approach, as well as cell-cell adhesion assays. Activation of signalling pathways in 2D and 3D cultures were performed by proteomic array and Western Blot analysis. RESULTS AND CONCLUSION GBM cells expressing Cav-1 (Cav-1 +ve) displayed a higher invasive capacity compared cells where Cav-1 had been silenced Cav-1 –ve), the latter also showing increased cell-cell adhesion. A significant finding from the signalling analysis was an inverse association between Cav-1 silencing and activation of AKT evidenced by increased phosphorylation at both Ser473 and Thr308 sites. Ongoing studies are exploring this signalling axis and its relationship to the invasive phenotype. CM and MG acknowledge Cancer Research Wales support. GP and HF acknowledge Brain Tumour Research support.

PKM2 promotes reductive glutamine metabolism.

Objective:Pyruvate kinases M (PKM), including the PKM1 and PKM2 isoforms, are critical factors in glucose metabolism. PKM2 promotes aerobic glycolysis, a phenomenon known as " the Warburg effect”. The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism.Methods:The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism. Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800. The metabolism of 13C6-glucose or 13C5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses. The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model. Results:We found that both PKM1 and PKM2 enabled aerobic glycolysis, but PKM2 converted glucose to lactate much more efficiently than PKM1. As a result, PKM2 reduced glucose levels reserved for intracellular utilization, particularly for the production of citrate, and thus increased the α-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetyl-coenzyme A through the reductive pathway. Furthermore, reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions, which supports in vivo tumor growth. In addition, PKM-deletion induced a reverse Warburg effect in tumor-associated stromal cells. Conclusions:PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis. This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.

Stromal Caveolin-1 and Caveolin-2 Expression in Primary Tumors and Lymph Node Metastases.

The expression of caveolin-1 (CAV1) in both tumor cell and cancer-associated fibroblasts (CAFs) has been found to correlate with tumor aggressiveness in different epithelial tumor entities, whereas less is known for caveolin-2 (CAV2). The aim of this study was to investigate the clinicopathological significance and prognostic value of stromal CAV1 and CAV2 expression in lung cancer. The expression of these two genes was investigated at protein level on a tissue microarray (TMA) consisting of 161 primary tumor samples. 50.7% of squamous cell lung cancer (SCC) tumors showed strong expression of CAV1 in the tumor-associated stromal cells, whereas only 15.1% of adenocarcinomas (AC) showed a strong CAV1 expression (p < 0.01). A strong CAV2 stromal expression was found in 46.0% of the lung tumor specimens, with no significant difference between the subtypes. Neither CAV1 nor CAV2 stromal expression was associated with any other clinicopathological factor including survival. When the stromal expression in matched primary tumors and lymph node metastases was compared, both CAV1 and CAV2 expressions were frequently found lost in the corresponding stroma of the lymph node metastasis (40.6%, p = 0.003 and 38.4%, p = 0.001, resp.). Loss of stromal CAV2 in the lymph node metastases was also significantly associated with earlier death (p = 0.011). In conclusion, in contrast to the expression patterns in the tumor tissue of lung cancer, stromal expression of CAV1 in primary tumors was not associated with clinical outcome whereas the stromal expression of especially CAV2 in the metastatic lymph nodes could be associated with lung cancer pathogenesis.

Abstract B115: MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts

Abstract Triple-negative breast cancer (TNBC) is a highly metastatic disease with very poor long-term prognosis. Because of the lack of well-defined molecular targets, traditional chemotherapy is currently the only therapeutic regimen for TNBC. Within this agent class, eribulin has been approved for metastatic breast cancer and the prespecified subgroup analysis in phase 3 clinical trial (study301) revealed that eribulin demonstrated the greatest therapeutic benefit on overall survival of TNBC patients among all other breast cancer subtypes. Folate receptor alpha (FRA) is a GPI-linked membrane glycoprotein that has limited expression in normal tissue, but is highly overexpressed on a large number of cancers of epithelial origin, including non-small cell lung cancer (NSCLC), ovarian cancer, and TNBC. These features make FRA an attractive target for the treatment of TNBC using targeted therapeutic approaches. Farletuzumab, a humanized monoclonal antibody targeting FRA, has been investigated in multiple clinical trials enrolling patients overexpressing FRA. MORAb-202, a novel antibody-drug conjugate (ADC) consisting of farletuzumab conjugated to eribulin, targets FRA overexpressed by a variety of tumor types including TNBC. Upon binding and internalization, it is hypothesized that the liberated eribulin elicits both a direct cytotoxic effect on FRA-positive tumor cells and a bystander effect on FRA-negative tumor cells and, importantly, tumor-associated stromal cells within the tumor microenvironment. To address this hypothesis, we have evaluated the in vivo efficacy of MORAb-202 against a FRA-positive TNBC patient-derived xenograft (PDX) model. Single administration of 5 mg/kg MORAb-202 mediated tumor regression statistically compared to vehicle control (P&amp;lt;0.0001). Additionally, one animal treated with MORAb-202 showed complete and durable tumor regression. In order to examine the effects of MORAb-202 treatment on the tumor microenvironment, we harvested tumors both prior to treatment and five days post-treatment. Immunofluorescent staining was used to assess the target-specific engagement of MORAb-202 to FRA-positive regions of the tumor. Strong membrane staining of MORAb-202 was observed, while no staining was seen in the adjacent stromal regions. MORAb-202 also had demonstrable effects on the tumor microenvironment via reduction of the number of FRA-negative cancer-associated fibroblasts (CAF) present. To further evaluate whether these effects on CAF are part of the mode of action (MOA) of MORAb-202, we tested the antitumor efficacy and effects on the tumor microenvironment in a surrogate FRA-positive in vivo model. Tumors were isolated on days 3, 5, 7, and 9 post-treatment. The reduction in CAF, visualized by alpha-smooth muscle actin staining, was directly proportional to overall tumor reduction. In both studies, single-dose MORAb-202 showed dramatically greater efficacy than molar equivalent dosing of eribulin alone. Histologic analyses revealed unique modes of action of MORAb-202 over antibody or payload alone. These results suggest a potential benefit of MORAb-202 in the treatment of TNBC. Citation Format: Katherine A. Rybinski, Christopher Maddage, Xin Cheng, Earl Albone, Luigi Grasso, Keiji Furuuchi, Toshimitsu Uenaka. MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B115.