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The role of the immune system during cancer development is complex involving extensive reciprocal interactions between genetically altered cells, adaptive and innate immune cells, their soluble mediators and structural components present in the neoplastic microenvironment. Each stage of cancer development is regulated uniquely by the immune system; whereas full activation of adaptive immune cells at the tumor stage may result in eradication of malignant cells, chronic activation of innate immune cells at sites of premalignant growth may actually enhance tumor development. In addition, the balance between desirable antitumor immune responses and undesirable pro-tumor chronic inflammatory responses largely depends on the context in which a malignancy is developing. The following chapter focuses on the inflammatory components and processes engaged during cancer development and the impact of the inflammatory microenvironment.

The main function of the mammalian immune system is to monitor tissue homeostasis, to protect against invading or infectious pathogens and to eliminate damaged cells. Therefore, it is surprising that cancer occurs with such a high frequency in humans. Recent insights that have been gained from clinical studies and experimental mouse models of carcinogenesis expand our understanding of the complex relationship between immune cells and developing tumours. Here, we examine the paradoxical role of adaptive and innate leukocytes as crucial regulators of cancer development and highlight recent insights that have been gained by manipulating immune responses in mouse models of de novo and spontaneous tumorigenesis.

The role of the immune system during cancer development is complex involving extensive reciprocal interactions between genetically altered cells, adaptive and innate immune cells, their soluble medi

Contains fulltext : 50884.pdf (Publisher’s version ) (Closed access) Tumours are complex tissues composed of ever-evolving neoplastic cells, matrix proteins that provide structural support and sequester biologically active molecules, and a cellular stromal component. Reciprocal interactions between neoplastic cells, activated host cells and the dynamic micro-environment in which they live enables tumour growth and dissemination. It has become evident that early and persistent inflammatory responses observed in or around developing neoplasms regulates many aspects of tumour development (matrix remodelling, angiogenesis, malignant potential) by providing diverse mediators implicated in maintaining tissue homeostasis, e.g., soluble growth and survival factors, matrix remodelling enzymes, reactive oxygen species and other bioactive molecules. This review highlights recent insights into the role of chronic inflammation associated with cancer development and examines proteolytic pathways activated by infiltrating leukocytes during neoplastic programming of tissues.

Tumour spread is the main cause of death in patients with melanoma. Exposure of melanoma to ultraviolet radiation has now been found to cause an inflammatory response that drives the formation of distant metastases. See Letter p.109 Ultraviolet (UV) radiation is known to induce melanoma, but whether and how UV irradiation influences the pathogenesis of melanoma indirectly through its effects on the microenvironment is unclear. Here Thomas Tuting and colleagues show that exposing mice to UV radiation in a manner that mimics mild sunburn can promote melanoma metastasis. It does so by inducing the release of the chromatin protein HMGB1 from damaged skin cells. HMGB1 induces inflammation, which in turn promotes angiogenesis and melanoma cell migration and metastasis formation. The authors observe that in this model melanoma, cells spread along the abluminal side of blood vessels in a process called angiotropism, which has been observed in patients, but so far not been mechanistically explained. These findings have important implications for the evaluation of UV exposure as a risk factor for melanoma.

Infiltration of leukocytes into premalignant tissue is a common feature of many epithelial neoplasms and is thought to contribute to cancer development. However, the molecular and cellular regulatory mechanisms underlying activation of innate host responses to enhanced neoplastic cell proliferation are largely unknown. Considering the importance of the complement system in regulating inflammation during acute pathologic tissue remodeling, we examined the functional significance of complement component 3 (C3) as a regulator of inflammatory cell infiltration and activation during malignant progression by using a transgenic mouse model of multistage epithelial carcinogenesis, e.g., HPV16 mice. Whereas abundant deposition of C3 is a characteristic feature of premalignant hyperplasias and dysplasias coincident with leukocyte infiltration in neoplastic tissue, genetic elimination of C3 neither affects inflammatory cell recruitment toward neoplastic skin nor impacts responding pathways downstream of inflammatory cell activation, e.g., keratinocyte hyperproliferation or angiogenesis. Taken together, these data suggest that complementindependent pathways are critical for leukocyte recruitment into neoplastic tissue and leukocytemediated potentiation of tumorigenesis.

Matrix metalloproteinases (MMPs) are a family of metalloendopeptidases that induce remodelling of extracellular matrix (ECM) and differentially cleave many soluble mediators that regulate cell physiology. Due to their matrix-degrading capabilities and elevated expression levels in both neoplastic and host cells in human cancer, MMPs have acquired considerable attention as targets for anti-cancer therapy. This chapter summarizes two decades of research examining MMP biochemistry and biology utilizing in vitro cell-based and biochemical analyses, more recent examination of their functional significance in de novo mouse models of human cancer development and results from human clinical trials where MMP inhibitors were evaluated for efficacy as anti-cancer therapeutics.

SummaryChronic inflammation predisposes tissue to cancer development; however, regulatory mechanisms underlying recruitment of innate leukocytes toward developing neoplasms are obscure. We report that genetic elimination of mature T and B lymphocytes in a transgenic mouse model of inflammation-associated de novo epithelial carcinogenesis, e.g., K14-HPV16 mice, limits neoplastic progression to development of epithelial hyperplasias that fail to recruit innate immune cells. Adoptive transfer of B lymphocytes or serum from HPV16 mice into T and B cell-deficient/HPV16 mice restores innate immune cell infiltration into premalignant tissue and reinstates necessary parameters for full malignancy, e.g., chronic inflammation, angiogenic vasculature, hyperproliferative epidermis. These findings support a model in which B lymphocytes are required for establishing chronic inflammatory states that promote de novo carcinogenesis.

MMPs are a family of at least 25 highly homologous secreted or plasma membrane-associated zinc-binding proteinases (reviewed in [1,2]). MMPs can be produced by virtually all cell types, and regulate many developmental processes and a variety of pathological conditions, including, but not limited to, cancer, auto-immune diseases, and periodontitis.

To provide protection against all foreign pathogens one can possibly encounter during their lifetime, the T cell repertoire has to be as diverse as possible. At the same time, it is desirable that the T cell repertoire remains unresponsive towards healthy tissues. To realize this self/nonself discriminatory property, T cells undergo tightly controlled selection processes during maturation in the thymus. The key parameter determining the outcome of these selection processes is the avidity of the T cells for self-MHC/self-peptide complexes expressed in the thymus; low avidity interactions result in positive selection, whereas high avidity interactions lead to negative selection. Despite the selection processes, self-tolerance is far from absolute. In many cases, this is due to the presence of self-antigen in the thymus at a level that is too low to induce thymic deletion. In addition, T cells with a low avidity for ubiquitously expressed self-antigens can escape clonal deletion and enter the periphery. A thorough understanding of the self-specific T cell repertoire is important because many potential targets for cancer immunotherapy are self-proteins. In this review, the authors discuss the impact of self-antigen expression on the CD8+ T cell repertoire. An overview of the fate and functional capacities of self-specific T cells with specificity for tissue-restricted self-antigens and for ubiquitously expressed self-antigens is provided. Furthermore, the authors discuss the influence of negative selection on the antitumor reactivity of the mature T cell repertoire.

Cancer-associated fibroblasts (CAFs) can promote breast cancer growth and metastasis, but the underlying mechanisms are still largely unknown. In order to study the role of CAFs in breast cancer development in an in vivo setting, we make use of a spontaneous mammary tumor mouse model for invasive lobular carcinoma (ILC). ILC is the second most common type of breast cancer, and is characterized by loss of E-cadherin, a rich stromal compartment, infiltrative growth, and treatment resistance. Often, the PI3K pathway is observed activated in the ILC subtype. Based on these characteristics, we have generated mouse models with mammary epithelial-specific conditional knockout of E-cadherin and PTEN that develop classical ILCs with a strong resemblance to human ILC. Using PDGFRβ as a fibroblast marker, we have observed an abundant presence of stromal fibroblasts in these ILCs, similar to human ILC. Based on a mammary gland transplantation setting using fluorescent labeled recipient mice, we have shown that fibroblasts get recruited from the host during tumor development. In vitro, we have also shown that tumor cells recruit fibroblasts. Inhibition of PDGFRβ in this in vitro model can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and cytokine profiling. We will subsequently assess their functional significance during ILC development and progression. This research is supported by a fellowship of the Dutch Cancer Society Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. The role of fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B77. doi:10.1158/1538-7445.CHTME14-B77

Over 90% of breast cancer deaths are due to complications as a consequence of metastasis formation. Much progress has been made in understanding primary breast cancer formation; however, metastatic disease is still largely unexplored, poorly understood and incurable. Clearly, there is an urgent need for novel therapies with efficacious anti-metastatic activity. The different steps of the metastatic cascade are largely regulated by reciprocal interactions between cancer cells and their microenvironment. Accumulating evidence indicates that cells and mediators of the immune system can facilitate metastasis formation. To mechanistically study how immune cells and their mediators modulate breast cancer metastasis, we have recently developed a mouse model of spontaneous breast cancer metastasis that mimics the clinical course of metastatic disease in humans. The basis is the K14cre;EcadF/F;p53F/F transgenic mouse that develops breast cancer resembling human invasive lobular carcinoma. We orthotopically transplant invasive lobular carcinoma fragments from these mice into mammary glands of wild-type syngeneic mice. Once primary tumors are established, we mimic the clinical setting and perform a mastectomy. Following surgery, the mice develop clinically overt metastases in lymph nodes, lungs, liver and other distant organs. This novel mouse model of breast cancer metastasis accurately mimics each step of the metastatic cascade in humans. It provides a unique tool to further explore the biology of metastatic breast cancer with the aim to contribute to the development of more effective treatment strategies. Neutrophils make up a significant proportion of the inflammatory infiltrate in many tumors and their accumulation in breast cancer patients has been associated with metastasis formation. Also in our spontaneous breast cancer metastasis mouse model, metastasis formation is accompanied by a very pronounced accumulation of neutrophils in circulation and distant organs. Antibody-mediated depletion of neutrophils did not affect primary breast cancer outgrowth, but did result in a profound decrease in lung and lymph node metastasis. Using biological and genomic approaches, we have uncovered a novel communication network between gamma delta T cells and neutrophils that is critical for breast cancer metastasis. We are currently dissecting the mechanisms by which neutrophils facilitate breast cancer metastasis formation. Besides regulating metastatic disease, the immune system also modulates responsiveness of cancer to conventional forms of therapy. Using the K14cre;EcdF/F;p53F/F mouse mammary tumor model, we study the ability of the immune system to influence the anti-cancer efficacy of chemotherapy. We have observed that it is very important to optimally match chemotherapeutic drugs with immunomodulatory compounds. In addition, combining chemotherapy with an immunomodulatory drug can trigger a rewiring of the inflammatory tumor microenvironment resulting in immune-dependent therapy resistance. Taken together, through mechanistic understanding of the crosstalk between the immune system and cancer, we aim to contribute to the design of novel immunomodulatory strategies to fight metastatic breast cancer and to increase the efficacy of conventional anti-cancer therapies. (Supported by the Dutch Cancer Society grant 2011-5004, NWO/VIDI 91796307, AICR 11-0677 and FP7 MCA-ITN 317445) Citation Format: Seth B. Coffelt, Chris W. Doornebal, Metamia Ciampricotti, Camilla Salvagno, Kelly Kersten, Kim Vrijland, Cheei-Sing Hau, Jos Jonkers, Karin E. De Visser. Cancer-associated inflammation facilitates metastatic breast cancer and counteracts chemoresponsiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA07. doi:10.1158/1538-7445.CHTME14-IA07

Can self-specific T cells that have escaped intrathymic deletion be exploited to generate antitumor immunity? To determine whether antitumor immunity to a self-Ag for which central tolerance exists can be generated, a mouse model is used in which a fragment of the influenza nucleoprotein (NP) is expressed as a transgene under the control of the H-2K promoter in C57BL/10 mice (B10NP mice). In these mice an oligoclonal population of NP-specific T cells escapes thymic and peripheral deletion and can be activated upon immunization. The main hallmark of these self-specific CD8+ T cells is diminished avidity for the pertinent MHC/peptide complex. We show in this study that intranasal infection with influenza virus can stimulate low-avidity NP-specific T cells to recognize and destroy NP-expressing microtumors in the lung, but not NP-expressing tumors growing s.c. Only a memory NP-specific CD8+ T cell response can suppress the growth of an s.c. growing NP-expressing tumor. This delay in tumor growth is associated with a dramatic increase in the number of circulating NP-specific CD8+ T cells. In addition, cultured memory NP-specific T cells require ∼100-fold less Ag to induce NP-specific lysis than primary T cells, consistent with the observation that memory T cells have an increased avidity due to affinity maturation. Finally, during an NP-specific memory response, substantial numbers of low-avidity NP-specific T cells can be recovered from s.c. growing tumors. Together, these findings indicate that, when only a low-avidity repertoire is available to generate antitumor immunity, the best strategy may be to enhance memory responses.

Pharmacological studies showed that an inhibitor (pertussis toxin) or activator (mastoparan) of the Ga-subunit of heterotrimeric GTP binding proteins can counteract the effect of the plant hormone abscisic acid (ABA) in the inhibition of barley embryo germination. The presence of several GTP binding proteins (ranging from 15 to 48 kDa) in membrane fractions of barley embryos was demonstrated by specific [a- 32 P]GTP labeling of proteins and by Western blotting using antibodies against highly conserved G protein a-subunit or Ras protein. GTP binding was enhanced in protein fractions from ABA-treated embryos, especially concerning poly-peptides of Mr 42, 29, 28, 27, and 25 kDa. Our data suggest that GTP-binding proteins are involved ABA-induced responses in barley embryo. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Increasing evidence from epidemiological and pathology studies indicates a role of the immune system in initiation and progression of multiple cancers, including prostate cancer. However, reports on the specific role of the immune system are contradictive since both suppression and acceleration of disease progression have been described. Overexpression of MYC and loss of PTEN are frequent genetic lesions in prostate cancer. It has been shown that these lesions induce chemokine expression, attraction of immune cells and subsequent angiogenesis and disease progression in cancer models. In this study we address the role of the adaptive immune system in prostate cancer progression in two independent spontaneous prostate cancer mouse models; the FVB/HiMYC mouse model in which prostate cancer formation is driven by transgenic expression of human MYC under control of the ARR2/probasin promoter, and the PB-Cre;PTENF/F mouse model in which prostate cancer formation is initiated by loss of PTEN expression. Both mouse models develop Prostatic Intraepithelial Neoplasia (PIN) from the age of 4 weeks on. FVB/HiMYC mice develop invasive carcinoma from the age of 24 weeks and PB-Cre;PTENF/F mice from the age of 12 weeks on. To address the functional significance of lymphocytes in prostate cancer development, FVB/HiMYC and PB-Cre;PTENF/F were crossed to lymphocyte deficient RAG-1−/− mice. Preliminary data suggest that both mouse models crossed to RAG-1−/− have a longer latency of invasive carcinoma development and less extensive lesions than RAG-1+/− mice, suggesting an important role of the adaptive immune system in prostate cancer initiation and/or progression. Marked inflammation and increased nuclear BrdU incorporation was found in concert with prostate cancer development using immunohistochemistry. Flow cytometry confirmed an increase in CD45 positive cells in the prostate, which was not observed in blood. Studies to identify the lymphocyte population with a key role in prostate cancer development and to dissect the exact mechanism of growth promotion are currently ongoing. In addition, we will verify whether the identified immune cells and soluble mediators in the in vivo models are also found in human prostate cancer samples. In conclusion: Our study provides insight into the role of the adaptive immune system in prostate cancer development. These data might serve as a basis to develop intervention strategies that intersect with the supportive role of the immune system in human prostate carcinogenesis. Citation Format: Monique H.M Melis, Johan van Burgsteden, Hester J.T. van Zeeburg, John Zevenhoven, Ji-Ying Song, Karin E. de Visser, Andre M. Bergman. The role of the adaptive immune system in initiation and progression in two independent spontaneous mouse models for prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2013-1268

We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing RAB mRNA expression was measured. In barley aleurone protoplasts, RAB gene expression was inhibited by blockers of the calcium channel and outward rectifying potassium channel (verapamil and bepridil). The inhibitory effects were dependent on the concentrations of blockers applied. The possible role of calcium in ABA-regulated gene expression in aleurone protoplasts was further analyzed by using calmodulin inhibitors and the Ca2+ antagonists Cd2+ and La3+, which showed a strong inhibition on RAB gene expression. In addition, indirect manipulation of the inositol cycle by using Li+ showed a dramatic stimulation of ABA-induced gene expression. Introducing inositol [1,4,5]-trisphosphate (lnsP3) into protoplasts had strong inhibitory effects on ABA-induced RAB gene expression, whilst addition of heparin could overcome this inhibitory effect. Based on these results, a possible role of calcium fluxes and intracellular calcium levels in ABA signal transduction is discussed.

After decades of research, breast cancer still remains the leading cause of cancer mortality in women. The majority of these breast cancer deaths is due to metastasis, which is still poorly understood and incurable. Recently it has been appreciated that the tumor microenvironment plays a significant role in both tumor progression and response to chemotherapy. However, its role in metastasis formation is still largely unclear. In this project we focus on the role of the inflammatory microenvironment on metastatic breast cancer and chemotherapy responsiveness. In our research we make use of the conditional K14cre;EcdF/F;P53F/F breast cancer mouse model that spontaneously develops mammary tumors resembling human invasive lobular carcinomas (ILC). These tumors reside in an inflammatory microenvironment which is characterized by expression of inflammatory cytokines resulting in infiltration of large numbers of immune cells. About 50% of K14cre;EcdF/F;P53F/F animals develop micrometastases in distant organs like lung, lymph node and spleen, however they do not succumb to metastatic disease. We hypothesize that the inflammatory tumor microenvironment plays a role in breast cancer metastasis formation and possibly in chemotherapy efficacy. Exploiting the K14cre;EcdF/F;P53F/F model, mouse ILC (mILC) pieces are transplanted into the 4th mammary gland of wild type recipient mice. Tumors are allowed to grow to a specific size upon which a mastectomy is performed mimicking the clinical situation. Mice are sacrificed as soon as they show clinical signs of metastatic disease. This transplantable model allows us to study the effect of the microenvironment on breast cancer metastasis and chemotherapy responsiveness in two distinct settings: a neo-adjuvant setting (treatment targeting primary tumor growth) and an adjuvant setting (treatment after mastectomy specifically targeting metastatic outgrowth). Using this model we set out to study the effect of the inflammatory microenvironment in breast cancer metastasis formation and chemotherapy responsiveness. We found that neo-adjuvant chemotherapy treatment resulted in a significant delay of primary tumor growth. As a result, metastasis-free survival was significantly increased compared to controls. Also adjuvant chemotherapy treatment resulted in a survival benefit compared to controls. We are currently studying the effect of inflammatory cytokine inhibition on chemotherapy responsiveness and formation of distant metastases. Therefore we are analyzing different populations of immune cells in the inflammatory tumor microenvironment by flow cytometry. Subsequently, we are looking into the pattern of metastasis formation throughout the body. This research is funded by the Dutch Cancer Society; 2011-5004. Citation Format: Kelly Kersten, Metamia Ciampricotti, Chris W. Doornebal, Cheei-Sing Hau, Seth B. Coffelt, Karin de Visser. The impact of the inflammatory microenvironment on breast cancer metastasis and chemotherapy responsiveness. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A96.

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.