Kelly Kersten

IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis

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)-1β elicits IL-17 expression from gamma delta (γδ) 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 γδ T cells prevents neutrophil accumulation and downregulates the T-cell-suppressive phenotype of neutrophils. Moreover, the absence of γδ 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 γδ T cell/IL-17/neutrophil axis—represents a new strategy to inhibit metastatic disease.

Genetically engineered mouse models in oncology research and cancer medicine

Genetically engineered mouse models (GEMMs) have contributed significantly to the field of cancer research. In contrast to cancer cell inoculation models, GEMMs develop de novo tumors in a natural immune‐proficient microenvironment. Tumors arising in advanced GEMMs closely mimic the histopathological and molecular features of their human counterparts, display genetic heterogeneity, and are able to spontaneously progress toward metastatic disease. As such, GEMMs are generally superior to cancer cell inoculation models, which show no or limited heterogeneity and are often metastatic from the start. Given that GEMMs capture both tumor cell‐intrinsic and cell‐extrinsic factors that drive de novo tumor initiation and progression toward metastatic disease, these models are indispensable for preclinical research. GEMMs have successfully been used to validate candidate cancer genes and drug targets, assess therapy efficacy, dissect the impact of the tumor microenvironment, and evaluate mechanisms of drug resistance. In vivo validation of candidate cancer genes and therapeutic targets is further accelerated by recent advances in genetic engineering that enable fast‐track generation and fine‐tuning of GEMMs to more closely resemble human patients. In addition, aligning preclinical tumor intervention studies in advanced GEMMs with clinical studies in patients is expected to accelerate the development of novel therapeutic strategies and their translation into the clinic.

exploiting the immunomodulatory properties of chemotherapeutic drugs to improve the success of cancer immunotherapy

Cancer immunotherapy is gaining momentum in the clinic. The current challenge is to understand why a proportion of cancer patients do not respond to cancer immunotherapy, and how this can be translated into the rational design of combinatorial cancer immunotherapy strategies aimed at maximizing success of immunotherapy. Here, we discuss how tumors orchestrate an immunosuppressive microenvironment, which contributes to their escape from immune attack. Relieving the immunosuppressive networks in cancer patients is an attractive strategy to extend the clinical success of cancer immunotherapy. Since the clinical availability of drugs specifically targeting immunosuppressive cells or mediators is still limited, an alternative strategy is to use conventional chemotherapy drugs with immunomodulatory properties to improve cancer immunotherapy. We summarize the preclinical and clinical studies that illustrate how the anti-tumor T cell response can be enhanced by chemotherapy-induced relief of immunosuppressive networks. Treatment strategies aimed at combining chemotherapy-induced relief of immunosuppression and T cell-boosting checkpoint inhibitors provide an attractive and clinically feasible approach to overcome intrinsic and acquired resistance to cancer immunotherapy, and to extend the clinical success of cancer immunotherapy.

Mammary tumor-derived CCL2 enhances pro-metastatic systemic inflammation through upregulation of IL1β in tumor-associated macrophages

ABSTRACT 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.

Assessment of PD-L1 expression across breast cancer molecular subtypes, in relation to mutation rate, BRCA1-like status, tumor-infiltrating immune cells and survival

ABSTRACT To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2−, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2−, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2− tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10–0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.

Abstract 575: PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer

Background: PD-1 blockade has emerged as an effective treatment for a subset of cancer patients. Studies have shown that PD-L1 expression is associated with likelihood of response to PD-1 blockade. In order to select the right breast cancer patient for immunotherapy, characterization of the immune landscape of breast tumors is required. Therefore, we assessed PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in different breast tumor subtypes and the link with prognosis. We also sequenced a panel of genes to assess the mutational load in triple negative tumors (TNBC) and investigate the association with PD-L1 positive TILs. Material and methods: We analyzed 438 tumor samples from breast cancer patients of all ages treated between 1986 and 2007 with surgery, with or without adjuvant therapy. PD-L1 was stained using whole slide specimens (E1L3N® antibody) after methodological validation. Pathologists quantified TILs based on International TILs Working Group recommendations and scored PD-L1 based on the percentage of positive (tumor and/or immune) cells; as negative if 0%, positive if ≥1%, and high if >50%. Mutational load was assessed based on DNA kinome sequencing. Associations were measured by Cox/logistic regression model, including pathological variables. Multiplex imaging of 20 immune-infiltrated areas from four ER negative tumors were performed using the Vectra® system based on immunofluorescence staining panel of: CD4, CD68, CD8, FOXP3 and PD-L1. Results: PD-L1 expression and TILs were higher in ductal (compared with lobular), high grade and estrogen receptor (ER)-negative tumors (p 50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (>50%) showed an association with increased mutation load (p=0.019) and a trend for better DMFS (HR=0.41; 95%CI: 0.16-1.04) compared with tumors lacking TILs. Further characterization of PD-L1 positivity in the immune-infiltrated cells was conducted by a multiplex imaging analysis. Preliminary results indicated that PD-L1 is expressed in CD68+, CD4+, FOXP3+ and CD8+ immune-cells. Conclusion: Our findings suggest that PD-L1 positive TILs are associated with worse prognosis in ER-positive breast cancer and with better outcome in ER-negative group. In TNBC, high mutational load correlates with high PD-L1 positive TILs. Citation Format: Marcelo Sobral-Leite, Koen Van de Vijver, Magali Michaut, Hugo M. Horlings, Tesa M. Severson, Philip C. Schouten, Rianne van der Linden, Kelly Kersten, Anna Marie Mulligan, Nayana Weerasooriya, Joyce Sanders, Ashley Cimino-Mathews, Dennis Peters, Gerrit K. Hooijer, Erik Hooijberg, Annegien Broeks, Rene Bernards, Sabine Linn, Irene L. Andrulis, Marc J. van de Vijver, Lodewyk F. Wessels, Marleen Kok, Karin E. de Visser, Marjanka K. Schmidt. PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2017-575

Abstract IA04: Cancer-associated systemic inflammation facilitates breast cancer metastasis

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.

Abstract A20: Mammary tumor-derived CCL2 enhances pro-metastatic systemic inflammation through upregulation of macrophage-derived IL1beta

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.

Abstract IA07: Cancer-associated inflammation facilitates metastatic breast cancer and counteracts chemoresponsiveness

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;Ecad F/F ;p53 F/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;Ecd F/F ;p53 F/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

Abstract A083: Neutrophils promote metastasis of invasive lobular carcinoma

The multistep process of metastasis is highly dependent on the intrinsic mutations that accumulate in cancer cells. However, recent experimental evidence has shown that these mutations are not sufficient to facilitate metastatic disease. Cues from the tumor microenvironment are also required. In order to understand how systemic changes might affect metastasis formation, we profiled the cellular and molecular alterations in various organs of K14cre;EcdF/F;p53F/F mammary tumor-bearing mice – a model of invasive lobular breast cancer. Among all immune cells, increased neutrophil accumulation was the most pronounced. To test the functional importance of neutrophils in the metastatic process, we utilized our recently described model of spontaneous metastasis. This model is based on the transplantation of K14cre;EcdF/F;p53F/F tumor pieces into wild-type recipient mice, and subsequent surgical removal of the primary tumor, permitting us to investigate neutrophil function in both the neoadjuvant and adjuvant settings. Interestingly, mice treated with neutrophil-depleting antibodies in the neoadjuvant setting displayed a profound reduction in pulmonary metastasis, whereas metastases in the adjuvant group were equivalent to controls. These data indicate that neutrophils play a prominent role during the very early stages of the metastatic cascade. Our preliminary data suggest that these tumor-educated neutrophils can suppress the activation of T cells, as one mechanism by which they may promote metastasis. In addition, cytokine analysis of K14cre;EcdF/F;p53F/F mammary tumors demonstrated an increase in the IL17 pathway when compared to wild-type mammary glands. Subsets of T cells are known producers of IL17 and this pathway regulates neutrophil expansion indirectly. In accordance with this, K14cre;EcdF/F;p53F/F mice lacking the adaptive immune system displayed reduced IL17 levels and reduced circulating neutrophils. Current efforts are underway to determine whether T cells play a role in the pro-metastatic function of neutrophils. Together, these findings indicate that neutrophils are crucial for the establishment of breast cancer metastasis. Citation Format: Seth B. Coffelt, Chris W. Doornebal, Kelly Kersten, Metamia Ciampricotti, Cheei-Sing Hau, Kim Vrijland, Jorieke Weiden, Jos Jonkers, Karin E. de Visser. Neutrophils promote metastasis of invasive lobular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A083.