유방암세포유래 엑소좀의 대식세포 및 수지상세포 면역조절 효과에 관한 연구

학위논문(박사)--서울대학교 대학원 :의과대학 의과학과,2020. 2. 문우경.서론: 암세포 유래 엑소좀은 면역세포와의 정보교환을 통해 종양면역을 조절하여 암의 진행과 전이에 관여한다고 알려져 있지만 유방암에서 엑소좀의 자극을 받은 대식세포 또는 수지상세포의 면역기능 변화가 암 진행과 전이에 주는 영향에 관한 생체 연구는 부족하다. 따라서 본 연구에서는 세포추적 영상으로 유방암세포유래 엑소좀과 대식세포 또는 수지상세포와의 상호작용을 관찰하고 엑소좀에 의한 대식세포와 수지상세포의 종양면역 기능 변화를 분석하고자 한다. 또한 생체광학영상기법과 일체형 광음향 초음파 영상기법을 이용하여 엑소좀에 의한 유방암 진행과 전이 기전을 규명하고자 한다. 실험방법: 본 연구에서는 사람과 마우스 삼중음성유방암세포(MDA-MB-231과 4T1), 마우스 대식세포(Raw264.7) 그리고 마우스 수지상세포(DC2.4)를 사용하였다. 렌티바이러스 시스템을 이용해 엑소좀 특이 막단백질(CD63)과 적색형광단백질(RFP)를 재조합한 CD63/RFP를 발현하는 MDA-MB-231－CD63/RFP, 4T1－CD63/RFP, 루시퍼라제(firefly luciferase)와 녹색형광단백질(GFP)을 동시에 발현하는 MDA-MB-231－Luc/GFP, GFP를 발현하는 RAW264.7/GFP와 DC2.4/GFP를 수립하였다. MDA-MB-231－CD63/RFP 와 4T1－CD63/RFP에서 분비되는 적색형광단백질을 발현하는 엑소좀을 추출하여, 나노입자분석 기기(NanoSight)를 사용하여 크기를 측정하고, 웨스턴 블럿으로 엑소좀 특이 단백질의 발현을 확인하였다. 추출한 엑소좀의 세포간 이동을 공초점 현미경으로 추적 관찰하였으며, 세포증식 평가(MTT)와 트렌스웰 이동 분석법으로 엑소좀에 의한 세포들의 증식, 이동 및 침윤 능력을 분석하였고, 실시간 역전사중합효소연쇄반응법, 웨스턴 블럿과 유세포 분석으로 대식세포와 수지상세포의 면역기능을 분석하였다. 면역결핍마우스(BALB/c nude)의 유선지방조직에 MDA-MB-231-Luc/GFP를 이식하여 유방암모델을 만들었다. 생체발광영상으로 엑소좀에 의한 종양의 성장과 전이 과정을 추적 관찰하였고 일체형 광음향 초음파 영상으로 EGFR항체가 탑재된 골드나노로드(anti-EGFR-GNs)를 일차 종양에 주입하여 유방암 림프절 전이를 분석하였다. 또한 마우스 피하에 엑소좀으로 활성시킨 수지상세포를 골드나노로드(GNs)로 표지하여 주사하고 피하부터 액와림프절로의 이동을 일체형 광음향 초음파 영상기법으로 추적 관찰 하였다. 영상 분석 후 적출한 종양 및 림프절 조직에서 H&E염색과 면역염색을 수행하였다. 결과: 유방암세포, 대식세포 그리고 수지상세포내에서 유방암세포 유래 엑소좀의 포식을 확인하였고 그 중 대식세포와 수지상세포는 엑소좀에 의해 활성화 되는 것이 관찰되었다. 엑소좀에 의해 각 세포들의 성장, 이동 및 침윤 능력이 증가되었고, 대식세포에서는 M1, M2 분극화 발현 마커인 NOS2와 CD206, Arginase-1의 발현이 유도되었고, 수지상세포에서는 보조자극 분자인 CD40, CD80, CD86의 발현과 이동성 관련 케모카인 수용체인 CCR7의 발현뿐만 아니라 분화와 성숙을 자극하는 TNF-α의 발현이 증가되었다. 생체발광영상과 일체형 광음향 초음파 영상으로 유방암 모델에서 엑소좀에 의해 종양의 성장과 전이가 촉진되는 것을 확인하였고 마우스의 피하에 이식한 엑소좀으로 활성 된 수지상세포는 림프절로의 이동 능력이 증가되었다. 결론: 형광단백질을 발현하는 유방암세포 유래의 엑소좀이 유방암세포, 대식세포, 수지상세포로 이동하는 과정을 실시간으로 추적 관찰한 결과, 엑소좀은 대식세포의 분극화와 수지상세포의 분화를 유도하여 유방암의 성장과 전이를 조절할 것이라는 가능성을 제시하였다. 또한 생체광학영상기법과 일체형 광음향 초음파 영상기법은 엑소좀에 의한 국소 림프절 전이 여부와 수지상세포의 이동과 분포를 효율적으로 추적 관찰하고, 분석할 수 있는 생체 영상법으로 다양한 암종에 활용에 활용될 수 있을 것이다.Introduction: Cancer cell-derived exosomes known as the mediators of intercellular communications, are involved in tumor progress and metastasis by modulating tumor immunity. Nevertheless, the role of macrophages or dendritic cells stimulated by breast cancer cell-derived exosome in breast tumor progression and metastasis is not fully elucidated. In this study, we investigated the interaction of breast cancer cell-derived exosomes with macrophages or dendritic cells, and analyzed the tumor immunity of macrophage and dendritic cells which stimulated by exosomes. We also analyzed the molecular mechanisms of exosome mediated breast cancer growth and metastasis using bioluminescent imaging and ultrasound-guided photoacoustic imaging. Methods: The human and mouse triple negative breast cancer cell lines MDA-MB-231 and 4T1, mouse macrophage cell line Raw264.7 and mouse dendritic cell line DC2.4 were used. MDA-MB-231－CD63/RFP, 4T1－CD63/RFP cells were established using lentivirus expressing RFP-tagged CD63, which is a specific marker of exosomes. MDA-MB-231－Luc/GFP cells expressing both green fluorescence protein (GFP) and the firefly luciferase (Luc), RAW264.7/GFP cells and DC2.4/GFP cells expressing GFP were established using a lentiviral system. RFP-tagged exosomes secreted from MDA-MB-231－CD63/RFP or 4T1－CD63/RFP cells were isolated. The size of isolated exosomes was measured by NanoSight and specific exosomal marker proteins were assessed by Western blot. The intercellular transfer of RFP tagged exosomes between cells was monitored by confocal microscopy. The effects of exosomes on cell proliferation, migration and invasive abilities were evaluated by MTT and Trans-well migration assays. The expression of genes associated with the anti-or pro-tumor immunity in the exosome-stimulated macrophage and dendritic cells were evaluated using RT-PCR, Western blot and flow cytometry. The xenograft tumor models were produced by injection with MDA-MB-231-Luc/GFP cells into the mammary gland of female Balb/c nude mice. Tumor progression and metastasis affected by exosomes were noninvasively monitored by bioluminescence imaging and ultrasound-guided photoacoustic imaging after intratumoral injection of anti-EGFR-GNs. Histologic examination of tissue was performed by H&E staining and immunostaining. Result: RFP tagged exosomes taken up by breast cancer cells, macrophages and dendritic cells can be tracked by live-cell microscopy, macrophage and dendritic cells when took up exosomes were exhibited slightly elongated morphology. Exosomes significantly increased cell growth, migration and invasion abilities. Exosomes activated macrophages to M1 and M2 phenotypes, which exhibited high expression of M1 marker (NOS2) and M2 markers (CD206, Arginase-1). Exosomes led to dendritic cell activation by inducing upregulation of co-stimulatory molecules CD40, CD80, and CD86, the chemokine receptor CCR7, and TNF-α. Primary tumor growth and metastasis promoted by exosomes and exosome-stimulated dendritic cell migration into lymph node were observed by bioluminescent imaging and ultrasound-guided photoacoustic imaging. Conclusions: We monitored the interaction of RFP-tagged exosomes with macrophages and dendritic cells in real-time. Our data demonstrated breast cancer cell-derived exosomes mediate breast cancer progress by modulating the immune function of macrophage and dendritic cells. The bioluminescent imaging and ultrasound-guided photoacoustic imaging combined with GNs allowed for sensitive and longitudinal monitoring of tumor growth, metastasis and dendritic cell migration in vivo.Chapter 1 1 Introduction 2 Materials and methods 5 Results 15 Discussion 25 Chapter 2 31 Introduction 32 Materials and methods 35 Results 48 Discussion 54 References 71 Abstract in korean 84Docto

CarboxyAmido-Triazole Orotate inhibits the growth of Imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

Chronic myelogenous leukemia is a myeloproliferative disorder characterized by the t(9:22) (q34:q11) reciprocal chromosomal translocation, resulting in the expression of the chimeric Bcr–Abl oncoprotein with constitutive tyrosine kinase activity. Deregulated Bcr–Abl induces the hyperactivation of various signalling pathways that promote cell growth, suppress apoptosis and alter cell adhesion. Bcr-Abl has also been involved in VEGF-mediated angiogenesis in CML and evidence indicates that the formation of new vessels plays an important role in the development and progression of CML. Imatinib mesylate (IM) is a selective well tolerated inhibitor of the Bcr–Abl tyrosine kinase that has significantly improved the prognosis of patients with chronic phase CML. Despite this remarkable progress, a major problem associated with the administration of imatinib is acquired resistance. Bcr-Abl gene amplification, increased expression of Bcr–Abl protein, point mutations in the Bcr–Abl tyrosine kinase domain have been reported as mechanisms of resistance to imatinib. Therefore, there is an urgent need for new anticancer agents and combinations that could improve responses and survival rates for CML. Recent studies from our laboratory have shown that addition of carboxyamidotriazole (CAI), an inhibitor of calcium-mediated signal transduction, to imatinib resistant human CML cells induces a marked decrease in cell viability and augmented apoptosis, events associated with downregulation of Bcr–Abl protein and inhibition of tyrosine phosphorylation of Bcr–Abl, STAT5, CrkL. Carboxyamidotriazole Orotate (CTO), is a derivate of CAI that has been developed at Tactical Therapeutics. CTO has a higher bioavailability and efficacy with respect to the parental compound. Exosomes are small vesicles of 40-100 nm diameter that are initially formed within the endosomal compartment and are secreted when a multivesicular body (MVB) fuses with the plasma membrane. These vesicles are released by many cell types including cancer cells and are considered messengers in intercellular communication. The exact function of exosomes in malignant cells has yet to be elucidated, but investigation has suggested roles in cell-to-cell communication, tumor-stroma interaction, and antigen presentation, thus potentially affecting cancer progression at different steps. Recent studies from our laboratory suggest that exosomes released from IM-sensitive CML cells directly affect endothelial cells modulating the process of neovascularization. Our data show that CTO is able to inhibit both in vitro and in vivo the growth of imatinib-resistant CML cells and to affect tumor microenvironment by modulating exosome-stimulated angiogenesis. CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility in the treatment of leukemia patients

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

The present study is designed to assess if exosomes released from Chronic Myelogenous Leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM-1 and VCAM-1 cell adhesion molecules and interleukin-8 expression. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE-cadherin and β-catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization

Serum-derived extracellular vesicles from breast cancer patients contribute to differential regulation of T-cell-mediated immune-escape mechanisms in breast cancer subtypes

BackgroundIntracellular communication within the tumour is complex and extracellular vesicles (EVs) have been identified as major contributing factors for the cell-to-cell communication in the local and distant tumour environments. Here, we examine the differential effects of breast cancer (BC) subtype-specific patient serum and cell-line derived EVs in the regulation of T cell mediated immune responses. MethodsUltracentrifugation was used to isolate EVs from sera of 63 BC patients, 15 healthy volunteers and 4 human breast cancer cell lines. Longitudinal blood draws for EV isolation for patients on neoadjuvant chemotherapy was also performed. Characterization of EVs was performed by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM) and immunoblotting. CD63 staining was performed on a tissue microarray of 218 BC patients. In-house bioinformatics algorithms were utilized for the computation of EV associated expression scores within The Cancer Genome Atlas (TCGA) and correlated with tumour infiltrating lymphocyte (TIL) scores. In vitro stimulation of PBMCs with EVs from serum and cell-line derived EVs was performed and changes in the immune phenotypes characterized by flow cytometry. Cytokine profiles were assessed using a 105-plex immunoassay or IL10 ELISA. ResultsPatients with triple negative breast cancers (TNBCs) exhibited the lowest number of EVs in the sera; whilst the highest was detected in ER+HER2+ cancers; reflected also in the higher level of CD63+ vesicles found within the ER+HER2+ local tumour microenvironment. Transcriptomic analysis of the TCGA data identified that samples assigned with lower EV scores had significantly higher abundance of CD4+ memory activated T cells, T follicular cells and CD8 T cells, plasma, and memory B cells; whilst samples with high EV scores were more enriched for anti-inflammatory M2 macrophages and mast cells. A negative correlation between EV expression scores and stromal TIL counts was also observed. In vitro experiments confirmed that circulating EVs within breast cancer subtypes have functionally differing immunomodulatory capabilities, with EVs from patients with the most aggressive breast cancer subtype (TNBCs) demonstrating the most immune-suppressive phenotype (decreased CD3+HLA-DR+ but increased CD3+PD-L1 T cells, increased CD4+CD127-CD25hi T regulatory cells with associated increase in IL10 cytokine production). In depth assessment of the cytokine modulation triggered by the serum/cell line derived exosomes confirmed differential inflammatory cytokine profiles across differing breast cancer subtypes. Studies using the MDA-231 TNBC breast cancer cell-line derived EVs provided further support that TNBC EVs induced the most immunosuppressive response within PBMCs.DiscussionOur study supports further investigations into how tumour derived EVs are a mechanism that cancers can exploit to promote immune suppression; and breast cancer subtypes produce EVs with differing immunomodulatory capabilities. Understanding the intracellular/extracellular pathways implicated in alteration from active to suppressed immune may provide a promising way forward for restoring immune competence in specific breast cancer patient populations

Trypanosoma brucei effect on leukocyte differentiation and activation

Tripanossomíase Africana ou doença do sono é uma doença zoonótica causada por Trypanosoma brucei, um protozoário parasita transmitido pela mosca tsetse ou Glossina. A introdução do parasita no hospedeiro vertebrado provoca uma sucessão de eventos que envolvem a imunidade inata e adaptativa. Os macrófagos (MΦ) apresentam um papel fundamental na defesa inata por serem células apresentadoras profissionais de antigénio (APC) e células fagocíticas, importantes na eliminação dos tripanossomas. A imunidade adaptativa é assegurada pelos linfócitos, nomeadamente pelos linfócitos T. Contudo, o papel desempenhado por estas células imunitárias ainda não se encontra completamente clarificado. Assim, este estudo teve como objetivo analisar a atividade dos MΦ na infeção por T. brucei, avaliando os níveis de ureia e óxido nítrico (NO) e a expressão membranar das moléculas de classe I (MHCI) e classe II (MHCII) do complexo principal de histocompatibilidade. A diferenciação das populações linfocitárias, T helper (Th), T citotóxicas (Tc) e T reguladoras foi também avaliada. MΦ de murganho foram expostos a tripomastigotas de T. brucei e estimulados com antigénio total e exossomas de T. brucei. A caracterização da ativação macrofágica através de ensaios colorimétricos demonstrou que ambos os fenótipos M1 e M2 foram expressos e foi evidenciada correlação positiva entre a produção de ureia e de NO. Adicionalmente, os resultados da citometria de fluxo indicaram que o parasita prejudica a diferenciação das subpopulações de MΦMHCI+ e MΦMHCII+ mas induz o aumento de moléculas MHCI. Contrariamente, os exossomas demonstraram estimular as funções APC através das moléculas MHCI e MHCII. Os rácios MHCI/MHCII indicaram que o contacto com o parasita favorece a apresentação antigénica às células TCD4+, enquanto que os exossomas direcionam a apresentação antigénica para as subpopulações de linfócitos TCD4+ e TCD8+. A caraterização das populações linfocitárias através da citometria de fluxo demonstrou que T. brucei causa a diminuição das populações de linfócitos Th e Tc. Contrariamente, a estimulação com o antigénio total ou com os exossomas induziram a expansão de ambas as subpopulações linfocitárias. T. brucei parece promover a expansão das subpopulações linfocitárias CD8-CD25+FoxP3- e CD8+CD25+FoxP3-. A expansão da subpopulação celular CD25-FoxP3+ foi observada na fração celular CD8- após estimulação do antigénio e na fração celular CD8+ estimulada por exossomas. Além disso, os exossomas também promoveram a expansão da subpopulação de linfócitos T CD8+CD25+FoxP3+. Curiosamente, apesar da contração celular T. brucei induziu o aumento das moléculas FoxP3. Em conjunto, os resultados obtidos indicam que o parasita e os exossomas parecem exercer efeitos opostos nas células analisadas. Os parasitas parecem minimizar a apresentação antigénica e evitam induzir a expansão das subpopulações de linfócitos T, facilitando a sua permanência no hospedeiro. Por outro lado, os exossomas segregados por T. brucei parecem estimular a apresentação antigénica e mediar a expansão dos linfócitos Th, possivelmente deslocando o foco da atividade do sistema imunitário dos parasitas para os exossomas. Estes resultados permitiram clarificar alguns dos princípios subjacentes à resposta imunitária inata e adaptativa na fase inicial da infeção. A compreensão destes mecanismos pode vir a contribuir para o desenvolvimento de novas estratégias de controlo e eliminação da Tripanossomíase Africana.African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by tsetse fly or Glossina. Parasite introduction into mammal hosts, triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defense, since they are antigen-presenting cells (APC) and have a phagocytosis function essential for trypanosomes clearance. Adaptive immune defense is carried out by lymphocytes, in particular by T lymphocytes. However, the exact role of these immune cells remains not completely understood. Thus, this study aimed to assess the role of MΦ in T. brucei infection by measuring the urea and nitric oxide (NO) levels, and by evaluating membrane expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T helper (Th), T cytotoxic (Tc) and T regulatory cell subsets was assessed. Mouse MΦ were exposed to T. brucei trypomastigotes and stimulated by T. brucei extract and T. brucei exosomes. Characterization of MΦ activation with colorimetric assays have indicated that both M1 and M2 phenotypes were expressed, evidencing a positive correlation between urea and NO levels produced. Additionally, results of flow cytometry indicated that T. brucei impairs the expansion of both MHCI+ and MHCII+ MΦ subsets, but enhanced MHCI molecules. On the contrary, T. brucei exosomes stimulated APC functions through MHCI and MHCII molecules. MHCI/MHCII rates indicated that T. brucei shift the antigen presentation to CD4+ T cells, while exosomes directed the antigen presentation to both CD4+ and CD8+ T cells. Characterization of lymphocyte subsets by flow cytometry demonstrated that T. brucei impairs both Th and Tc lymphocytes. On the contrary, cell stimulation by extract and exosomes promote the expansion of both T cell subpopulations. T. brucei seem to promote the expansion of CD8-CD25+FoxP3- and CD8+CD25+FoxP3- T cell subsets. The expansion of CD25-FoxP3+ T cell subset was observed in CD8- cell fraction antigen stimulated and in the CD8+ cell fraction exosome stimulated. Moreover, exosomes also induced the expansion of CD8+ CD25+FoxP3+ T cell subset. Interestingly, despite cell decrease T. brucei seemed to increase FoxP3 molecules. Taken together, these findings indicate that parasite and parasite exosomes seem to have opposite effects on the evaluated cells. Parasites seem to minimize antigen presentation and avoid inducing the expansion of T cell subsets, facilitating its permanence in the host. On the other hand, T. brucei secreted exosomes seems to induce APC functions and mediate the expansion of Th lymphocytes, probably focusing the immune activity on the exosomes and not on the parasites. These findings allowed to understand some underlying principles of the innate and adaptive immune response in the early-stage of infection. Comprehension of these mechanisms can endorse the development of new strategies for control and elimination of African Trypanosomiasis

Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through NF-Kβ dependent glycolytic metabolic reprogramming.

The formation of a pre-metastatic niche is a fundamental requirement for primary tumor metastasis. One of the defining characteristics of a pre-metastatic niche is infiltration of immunosuppressive macrophages. However, how these macrophages acquire their immunosuppressive phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDE) polarize macrophages towards an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent metabolic reprogramming in mice and humans. While NF-κB has previously been shown to act as a direct transcription factor for PD-L1, we report a novel mechanism where TDE-induced NF-κB activation drives PD-L1 expression by augmenting the glycolytic capacity of macrophages through two separate pathways. First, NF-κB increases glucose uptake into macrophages via a HIF-1α/GLUT-1-dependent mechanism. Secondly, elevated NOS2-dependent nitric oxide inhibits mitochondrial oxidative phosphorylation resulting in an increased conversion of pyruvate to lactate. Lactate then feeds back on NF-κB further increasing PD-L1 expression. Analysis of metastasis negative draining lymph nodes of non-small cell lung cancer patients revealed that macrophage PD-L1 expression positively correlates with expression levels of GLUT-1 and exosomal release genes YKT6 and TSG101 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link between exosomes, metabolism, and metastasis

Exosome-mediated crosstalk between chronic myelogenous leukemia cells and human bone marrow stromal cells triggers an interleukin 8-dependent survival of leukemia cells

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the Bcr–Abl oncoprotein with constitutive tyrosine kinase activity. Exosomes are nanovesicles released by cancer cells that are involved in cell-to-cell communication thus potentially affecting cancer progression. It is well known that bone marrow stromal microenvironment contributes to disease progression through the establishment of a bi-directional crosstalk with cancer cells. Our hypothesis is that exosomes could have a functional role in this crosstalk. Interleukin-8 (IL 8) is a proinflammatory chemokine that activates multiple signalling pathways downstream of two receptors (CXCR1 and CXCR2). We demonstrated that exosomes released from CML cells stimulate bone marrow stromal cells to produce IL 8 that, in turn, is able to modulate both in vitro and in vivo the leukemia cell malignant phenotype

Therapeutic targeting of tumor associated macrophages.

Previous studies on the mechanistic induction of anti-tumor responses by IL-12 cytokine therapy have focused on the adaptive immune response, specifically the activation NK cells and T cells as the primary targets of IL-12 treatment. In contrast, little attention has been given to the potential role of macrophages in the initiation of anti-tumor responses by IL-12 therapy despite reports that macrophages play a major role in promoting tumor growth and metastasis and in suppressing anti-tumor immune responses. Based on the functional adaptivity hypothesis, which is the concept that macrophages functionally adapt, rather than differentiate into specific mature subsets, in response to environmental stimuli, we hypothesized that tumor infiltrating (TIMs) as well as tumor-associated macrophages (TAMs) could be converted from tumor supportive activities to pro-immunogenic activities by IL-12 therapy. We examined the functional activities displayed by TIMs and TAMs after treatment with IL-12. Our data demonstrate (1) that tumor cells and tumor exosomes activate TIMs and TAMs by cell-contact dependent mechanisms involving ligation of CD40 and/or NKG2D, (2) that IL-12 treatment both in vivo and in vitro induces a rapid reduction of tumor supportive activities and a concomitant increase in pro-inflammatory activities in TIMs as well as TAMs, (3) that IL-12 induces a rapid release of cytoplasmic IL-I5 from the in situ activated tumor associated macrophages and (4) the release of IL-15 in essential to the recruitment of lymphocytes to the tumor and the metastatic lung, and to the destruction of the tumor and clearance of metastasis. It is concluded that macrophages in the tumor environment are activated and functionally modulated by the tumor. TIMs and TAMs respond to IL-12 treatment by rapidly converting from suppressive, tumor supportive activities to inflammatory, pro-immunogenic activities. Tumor associated macrophages thus are a critical target of IL-12 therapy and may orchestrate the subsequent NK and T cell cytotoxic response against the tumor

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells