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Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (T-regs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive T-regs during primary tumor growth. Tumor-educated T-regs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as T-reg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that T-regs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis, In line, an increased T-reg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ T cells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ T cell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1β, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1β and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.

Recent studies have revealed a role for macrophages and neutrophils in limiting chemotherapy efficacy; however, the mechanisms underlying the therapeutic benefit of myeloid-targeting agents in combination with chemotherapy are incompletely understood. Here, we show that targeting tumour-associated macrophages by colony-stimulating factor-1 receptor (CSF-1R) blockade in the K14cre;Cdh1F/F;Trp53F/F transgenic mouse model for breast cancer stimulates intratumoural type I interferon (IFN) signalling, which enhances the anticancer efficacy of platinum-based chemotherapeutics. Notably, anti-CSF-1R treatment also increased intratumoural expression of type I IFN-stimulated genes in patients with cancer, confirming that CSF-1R blockade is a powerful strategy to trigger an intratumoural type I IFN response. By inducing an inflamed, type I IFN-enriched tumour microenvironment and by further targeting immunosuppressive neutrophils during cisplatin therapy, antitumour immunity was activated in this poorly immunogenic breast cancer mouse model. These data illustrate the importance of breaching multiple layers of immunosuppression during cytotoxic therapy to successfully engage antitumour immunity in breast cancer.

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (T

Background Breast cancer (BrC) is the most prevalent cancer in women worldwide. Unfortunately, still limited treatment options are available for the most aggressive subtypes (i.e. hormone receptor [HR] negative). The response to neoadjuvant chemotherapy (NACT) in patients with HR-negative BrC can in part be influenced by an effective anti-tumor immune response. The sentinel lymph node (SLN) is the first site where BrC-specific T cell priming will occur but unfortunately it is also a major target of BrC-induced immune suppression. Lymph-node resident dendritic cells (LNR-DC) were found to be suppressed in metastatic SLN.1 In addition, this tumor-mediated immune suppression of LNR-DC is related to high-risk triple-negative BrC and may be a negative predictor for prognosis1. Preliminary data showed that NACT further reduced the activation status of LNR-DC. The goal of this study is to identify immune-enhancing agents that can counteract the tumor-mediated immune suppression of LNR-DC and promote tumor-specific T cell responses in order to improve therapy outcome in BrC patients upon NACT. Materials and Methods Phenotypic analyses were performed on immune-cell subsets in human BrC SLN using multi-color flow cytometry. In addition, ex-vivo cultures with human BrC SLN-derived cells and in vivo mouse experiments were performed to study the therapeutic efficacy of Toll-like receptor (TLR)-ligands (R848 and CpG) and a STING-ligand (STING-L; 2’3’-c-di-AM(PS)2(Rp,Rp)). Results Higher rates of LNR-DCs, but with an apparently reduced activation state, were found in SLN of NACT-treated patients compared to patients treated with surgery only. A comparative ex-vivo study with SLN cultures on the effects of R848, CpG-B and STING-L showed R848 to be superior in terms of LNR-DC activation. In a Krt14 (K14)-cre;Cdh1F/F;Trp53F/F (KEP) BrC mouse model, the effects of intratumoral administration of TLR- and STING-L were determined in combination with doxorubicin. STING-L outperformed R848 and CpG-B in terms of controlling primary tumor growth. Of note, in human ex-vivo cultures CpG-B proved effective in LNR-DC activation when combined with a STAT3 inhibitor, leading to the boosting of mammaglobin-specific T cell responses, Th1 skewing, and a drop in CpG-induced Treg levels. Conclusions In summary, intratumoral delivery of TLR- and STING-ligands in combination with NACT might be an interesting therapeutic approach in patients with high-risk HR-negative BrC, leading to SLN potentiation and enhanced antitumor T cell immunity. Future clinical studies should demonstrate the therapeutic benefit of this approach. Reference van Pul, et al. 2019, Journal for ImmunoTherapy of Cancer. Disclosure Information N. Prokopi: None. M. Heeren: None. I. Milenova: None. K. van Pul: None. T. Muijlwijk: None. M. Arends: None. S. van der Velde: None. K. Vrijland: None. A. van Weverwijk: None. K. de Visser: None. R. van de Ven: None. T. de Gruijl: None.

Microflora-induced TLR signaling in the intestinal epithelium is essential for a proper intestinal barrier function. Because of the close interactions of this epithelial layer with underlying mononuclear phagocytes, we hypothesized that epithelial TLR signaling may affect the differentiation of myeloid cell populations. In in vitro cultures we observed that colonic epithelial monolayers actively transported TLR2 ligands towards their basolateral side. The transported TLR2 ligands strongly stimulated the development of Ly6C(+) monocytes, while dendritic cell differentiation was inhibited. The TLR2 effect on monocyte and dendritic cell differentiation was mediated by the production of G-CSF. Mice lacking TLR signaling and mice that were treated with antibiotics showed decreased numbers of Ly6C(+) monocytes in bone marrow and spleen, which points to a role for microbial derived TLR-ligands in the homeostasis of Ly6C(+) monocytes. In conclusion, our results indicate that TLR ligands that are transported by intestinal epithelial cells stimulate Ly6C(+) monocyte development and suggest that this process may be involved in the maintenance of systemic Ly6C(+) monocyte numbers.

Patients with primary solid malignancies frequently exhibit signs of systemic inflammation. Notably, elevated levels of neutrophils and their associated soluble mediators are regularly observed in cancer patients, and correlate with reduced survival and increased metastasis formation. Recently, we demonstrated a mechanistic link between mammary tumor-induced IL17-producing γδ T cells, systemic expansion of immunosuppressive neutrophils and metastasis formation in a genetically engineered mouse model for invasive breast cancer. How tumors orchestrate this systemic inflammatory cascade to facilitate dissemination remains unclear. Here we show that activation of this cascade relies on CCL2-mediated induction of IL1β in tumor-associated macrophages. In line with these findings, expression of CCL2 positively correlates with IL1Β and macrophage markers in human breast tumors. We demonstrate that blockade of CCL2 in mammary tumor-bearing mice results in reduced IL17 production by γδ T cells, decreased neutrophil expansion and enhanced CD8+ T cell activity. These results highlight a new role for CCL2 in facilitating the breast cancer-induced pro-metastatic systemic inflammatory γδ T cell–IL17–neutrophil axis.

CD169-positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169-positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115+ F4/80(lo) CD11c(lo) macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll-like receptor signalling in mucosal homeostasis, the presence of CD169+ macrophages was not affected in mice that were deficient in MyD88-mediated Toll-like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169+ macrophages was dependent on lymphotoxin α, colonic CD169+ macrophages were present in normal numbers in lymphotoxin α-deficient mice. In contrast, reduced numbers of CD169+ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169+ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169+ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.

Introduction Breast cancer is the most frequent malignancy among women worldwide. More than 90% of breast cancer-related deaths are due to metastatic disease. Despite these facts, breast cancer metastasis remains incurable. A key player in metastasis is the immune system. Cancer-induced immunosuppression contributes to a tumour’s ability to evade immune destruction. A major cell type in this process is the regulatory CD4+ T cell (Treg). It has been reported that Tregs are found in the microenvironment of primary tumours and metastases, and that their depletion reduces metastatic burden; however, the underlying mechanisms remain poorly understood. Using state-of-the-art breast cancer mouse models that closely recapitulate primary human breast cancer and metastatic disease, our goal is to elucidate the impact of Tregs on breast cancer metastasis, focusing on differences between (pre-)metastatic tissues and different steps in the metastatic cascade. Material and methods To study primary spontaneous mammary tumours and the pre-metastatic niche, we primarily use the FVB K14cre;Cdh1F/F;Trp53F/F (KEP) conditional mouse model for invasive lobular carcinoma. For metastatic disease, KEP tumour fragments are orthotopically transplanted into in wild-type syngeneic FVB mice. Following tumour outgrowth and mastectomy, widespread metastatic disease is present in lungs, lymph nodes and other distant organs, providing an accurate representation of the different steps of the metastatic cascade. Results and discussions We observed systemically elevated levels of Tregs prior to metastatic disease. These tumor-educated Tregs display a distinct phenotype and specifically accumulate in the lung and lymph node metastases that arise in our metastasis models, perhaps indicating their possible importance for metastasis formation and progression. In addition to these systemic changes, within primary tumours and metastases a large population of PD1high Tregs is found. Preliminary data suggest that tumor-associated myeloid cells influence this population. Conclusion We are currently setting out to dissect the mechanism behind this interplay of intra-tumoral immune cells and the role of distinct Tregs found prior to metastatic disease.

The tumour-modulating effects of the endogenous adaptive immune system are rather paradoxical. Whereas some clinical and experimental observations offer compelling evidence for the existence of immunosurveillance, other studies have revealed promoting effects of the adaptive immune system on primary cancer development and metastatic disease. We examined the functional significance of the adaptive immune system as a regulator of spontaneous HER2(+) breast tumourigenesis and pulmonary metastasis formation, using the MMTV-NeuT mouse model in which mammary carcinogenesis is induced by transgenic expression of the activated HER2/neu oncogene. Although T and B lymphocytes infiltrate human and experimental HER2(+) breast tumours, genetic elimination of the adaptive immune system does not affect development of premalignant hyperplasias or primary breast cancers. In addition, we demonstrate that pulmonary metastasis formation in MMTV-NeuT mice is not dependent on the adaptive immune system. Thus, our findings reveal that spontaneous HER2-driven mammary tumourigenesis and metastasis formation are neither suppressed, nor altered by immunosurveillance mechanisms, nor promoted by the adaptive immune system.

Introduction Immunotherapies that target immune checkpoint molecules proved effective for the treatment of several solid tumours. However, the majority of breast cancer patients does not respond to this kind of approach, underlining the need of new strategies able to elicit an effective anti-tumoral immune response. The combination of conventional and immuno-based therapies represents a promising strategy to elicit such response. Our aim is to evaluate the efficacy of immunotherapy and chemotherapy combination in breast cancer, to dissect its effect on the endogenous anti-tumoral cytotoxic CD8 +T cells, and finally to assess the impact of a spontaneously developing tumour on T-cell biology. Material and methods The experiments are performed in K14cre;Cdh1F/F;Trp53F/F (KEP) mice, a spontaneous breast tumour model that closely resembles human invasive lobular carcinoma. Mice are treated with α-CTLA-4 and α-PD-1 antibodies, as a single treatment modality or in combination with MTD dosing Cisplatin or docetaxel. Therapy is initiated when the tumour has reached 50mm2 and continues until it reached 225mm2 or after a pre-defined time point, depending on the experiment. Results and discussions Mammary tumours in KEP mice show an altered T-cell balance compared to tumor-free mammary glands, with a reduced infiltration of CD8+ T cells and conventional CD4+ T cells, and higher percentage of T regulatory cells. Tumor-infiltrating CD8+ T cells (TILs) have an increased expression of multiple inhibitory receptors, and reduced IFNγ production, compared to peripheral CD8 +T cells of KEP mice. In vitro exposure of sorted CD8 +TILs to IL-15 or IL-2 augments their capacity to produce IFNγ, indicating that their suppressed state is reversible. Treatment of tumor-bearing KEP mice with a-CTLA-4 and a-PD-1 does not affect the tumour growth, but a synergistic therapeutic benefit is observed when α-CTLA-4 and α-PD-1 are combined with cisplatin, and not docetaxel, in a CD8+ T-cell dependent mechanism. Conclusion CD8 +TILs display some features of dysfunctional T cells. However, ex vivo cytokine stimulation can restore part of their effector functions, indicating that they are not terminally dysfunctional. Nonetheless, only the combination with chemotherapy and immunotherapy is able to elicit an efficient CD8 +T cell response, in a drug-dependent manner. We are currently dissecting the mechanisms underlying the synergistic therapy response, in order to ultimately contribute to the rational design of new immunomodulatory treatment strategies for breast cancer.

Thymidine phosphorylase (TP) catalyzes the phosphorylytic cleavage of thymidine to thymine and deoxyribose-1-phosphate. The latter may be involved in the angiogenic stimulation of TP. In the present study, we investigated whether thymidine and deoxyribose (dR) could stimulate angiogenesis in vitro of two types of endothelial cells (isolated from umbilical veins (HUVEC) and endothelial colony forming cells (ECFC)), and whether the stereoisomer L-deoxyribose (L-dR) and the thymidine phosphorylase inhibitor (TPI) could reduce this. Both cell types had a low TP activity. Thymidine increased the migration of both HUVECs and ECFCs, but dR only that of the ECFCs. The invasion was not changed by any of the agents tested. In conclusion, TP may play a role in the migration of HUVECs and ECFCs, but not the invasion.

Item does not contain fulltext Metastatic disease remains the primary cause of death for patients with breast cancer. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and their microenvironment. Within this local microenvironment and in distant organs, immune cells and their mediators are known to facilitate metastasis formation. However, the precise contribution of tumour-induced systemic inflammation to metastasis and the mechanisms regulating systemic inflammation are poorly understood. Here we show that tumours maximize their chance of metastasizing by evoking a systemic inflammatory cascade in mouse models of spontaneous breast cancer metastasis. We mechanistically demonstrate that interleukin (IL)-1beta elicits IL-17 expression from gamma delta (gammadelta) T cells, resulting in systemic, granulocyte colony-stimulating factor (G-CSF)-dependent expansion and polarization of neutrophils in mice bearing mammary tumours. Tumour-induced neutrophils acquire the ability to suppress cytotoxic T lymphocytes carrying the CD8 antigen, which limit the establishment of metastases. Neutralization of IL-17 or G-CSF and absence of gammadelta T cells prevents neutrophil accumulation and downregulates the T-cell-suppressive phenotype of neutrophils. Moreover, the absence of gammadelta T cells or neutrophils profoundly reduces pulmonary and lymph node metastases without influencing primary tumour progression. Our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune system--the gammadelta T cell/IL-17/neutrophil axis--represents a new strategy to inhibit metastatic disease.

Morphine and other opioid analgesics are potent pain-relieving agents routinely used for pain management in patients with cancer. However, these drugs have recently been associated with a worse relapse-free survival in patients with surgical cancer, thus suggesting that morphine adversely affects cancer progression and relapse. In this study, we evaluated the impact of morphine on breast cancer progression, metastatic dissemination, and outgrowth of minimal residual disease. Using preclinical mouse models for metastatic invasive lobular and HER2 breast cancer, we show that analgesic doses of morphine do not affect mammary tumor growth, angiogenesis, and the composition of tumor-infiltrating immune cells. Our studies further demonstrate that morphine, administered in the presence or absence of surgery-induced tissue damage, neither facilitates de novo metastatic dissemination nor promotes outgrowth of minimal residual disease after surgery. Together, these findings indicate that opioid analgesics can be used safely for perioperative pain management in patients with cancer and emphasize that current standards of "good clinical practice" should be maintained.

The administration of helminths is considered a promising strategy for the treatment of autoimmune diseases due to their immunomodulatory properties. Currently, the application of the helminth Trichuris suis as a treatment for Crohn's disease is being studied in large multi-center clinical trials. The intestinal epithelium forms an efficient barrier between the intestinal lumen containing the microbial flora and helminths, and dendritic cells (DCs) present in the lamina propria that determine the TH response. Here, we investigated how excreted/secreted (E/S) products of T. suis affect the barrier function of intestinal epithelial cells (IECs) in order to reach the DCs and modulate the immune response. We show that T. suis E/S products reduce the barrier function and the expression of the tight junction proteins EMP-1 and claudin-4 in IEC CMT93/69 monolayers in a glycan-dependent manner. This resulted in an increased passage of soluble compounds to the basolateral side that affected DC function. In addition, T. suis E/S suppressed LPS-induced pro-inflammatory cytokine production by CMT93/69 cells, whereas the production of the TH2 response-inducing cytokine thymic stromal lymphopoietin (TSLP) was induced. Our studies indicate that T. suis E/S glycans affect the function of the intestinal epithelium in order to modulate DC function. Identification of the T. suis E/S glycans that modulate IEC and DC function may lead to a strategy to reduce symptoms of autoimmune and allergic immune diseases by orally administrated helminth-derived factors without the need of infection with live helminths.

Over 90% of breast cancer deaths are due to complications as a consequence of metastasis formation. Despite its devastating effects, metastatic disease is still poorly understood. Accumulating evidence indicates that cells and mediators of the immune system influence metastasis formation. In our lab, we use preclinical mouse models for metastatic breast cancer to dissect the impact of the immune system on the different steps of the metastatic cascade. Neutrophils make up a significant proportion of the inflammatory infiltrate in many tumors and their systemic accumulation in cancer patients has been associated with metastasis formation. Also in our recently developed spontaneous breast cancer metastasis mouse model that accurately mimics each step of the metastatic cascade in humans, metastasis formation is accompanied by pronounced systemic accumulation of neutrophils. Antibody-mediated depletion of neutrophils did not affect primary breast cancer outgrowth, but profoundly decreased spontaneous metastasis formation in lungs and lymph nodes. Using biological and genetic approaches, we have uncovered a novel mammary tumor-induced systemic communication network between gamma delta T cells and neutrophils that is critical for breast cancer metastasis. Our data indicate that targeting this novel cancer cell-initiated systemic inflammatory cascade represents a viable strategy to inhibit metastatic disease. Citation Format: Seth B. Coffelt, Kelly Kersten, Max Wellenstein, Chris W. Doornebal, Camilla Salvagno, Kim Vrijland, Cheei-Sing Hau, Jos Jonkers, Karin E. de Visser. Cancer-associated systemic inflammation facilitates breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA04.

Metastatic breast cancer remains a major cause of cancer-related death among women. Metastasis is regulated by extensive crosstalk between cancer cells and immune cells. Besides the onset of a local inflammatory microenvironment, tumors frequently induce a systemic inflammatory state characterized by the release of various cytokines, chemokines and growth factors to mobilize myeloid cells that support metastasis, emphasizing the notion that cancer should be regarded as a systemic disease. Utilizing the K14cre;Cdh1F/F;Trp53F/F (KEP) conditional mouse model of metastatic breast cancer, we have recently unraveled a novel mechanistic link between mammary tumor-induced IL17-producing gamma delta T cells, systemic expansion of immunosuppressive neutrophils and metastasis formation. We identified tumor-derived IL1beta as one of the drivers of this cascade by inducing IL17 release from gamma delta T cells. However, it remains unclear how other inflammatory mediators are involved in this systemic inflammatory cascade. In the current study, we identified the pro-inflammatory cytokine CCL2 as a key regulator of the gamma delta T cell IL17 neutrophil axis. CCL2 blockade resulted in reduced IL17-production by gamma delta T cells, decreased systemic neutrophil accumulation and enhanced CD8+ T cell activity. We show that activation of this cascade relies on CCL2-mediated release of IL1beta from CCR2+ tumor-associated macrophages. These results indicate that CCL2, via IL1beta, acts as a key player in regulating the pro-metastatic gamma delta T cell IL17 neutrophil systemic inflammatory cascade and might be an interesting candidate for therapeutic targeting. Citation Format: Kelly Kersten, Seth B. Coffelt, Niels J. Verstegen, Kim Vrijland, Metamia Ciampricotti, Chris W. Doornebal, Cheei-Sing Hau, Parul Doshi, Karin E. de Visser. Mammary tumor-derived CCL2 enhances pro-metastatic systemic inflammation through upregulation of macrophage-derived IL1beta. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A20.

Many DNA vaccine candidates have been developed for the treatment of human papillomavirus type 16 (HPV16)-induced malignancies. Most of these vaccines consist of a fusion of E7 with a "carrier-protein" that functions to increase the potency of the vaccine. The nature of these carrier-proteins varies widely, and the mechanisms proposed to explain the enhanced immunogenicity of such fusions are often linked to the biological function of the carrier-protein. However, the potentiating effect of these carrier-proteins might also be explained by more general mechanisms, such as the provision of CD4+ T-cell help, increased antigen stability, or altered subcellular localization of the antigen. To assess whether these more generic mechanisms could suffice to generate highly immunogenic DNA vaccines, we evaluated a series of modular HPV16 E7 DNA vaccines in which the presence of CD4+ T-cell help, the presence of an endogenous carrier-protein, and the subcellular localization of the antigen could be systematically altered. Using this approach, we demonstrate that the addition of an element that provides CD4+ T-cell help, elements that enforce endoplasmic reticulum (ER) localization/retention are both necessary and sufficient to create markedly effective HPV16 E7-directed DNA vaccines. Importantly, the resulting design rules also apply to an HPV16 E6-directed DNA vaccine. The developed "HELP(ER)" HPV DNA vaccines encode only very limited additional sequences besides the antigen, thereby reducing the risk of antigenic competition and/or autoimmunity.

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