Metamia Ciampricotti

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.

Developmental stage-specific contribution of LGR5 + cells to basal and luminal epithelial lineages in the postnatal mammary gland

The leucine‐rich repeat‐containing heterotrimeric guanine nucleotide‐binding protein‐coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage‐tracing studies and mammary gland reconstitutions with LGR5‐expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage‐specific contribution of LGR5‐expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development. Copyright

Chemotherapy response of spontaneous mammary tumors is independent of the adaptive immune system.

Chemotherapy response of spontaneous mammary tumors is independent of the adaptive immune system

A Preclinical Mouse Model of Invasive Lobular Breast Cancer Metastasis

Metastatic disease accounts for more than 90% of cancer-related deaths, but the development of effective antimetastatic agents has been hampered by the paucity of clinically relevant preclinical models of human metastatic disease. Here, we report the development of a mouse model of spontaneous breast cancer metastasis, which recapitulates key events in its formation and clinical course. Specifically, using the conditional K14cre;Cdh1(F/F);Trp53(F/F) model of de novo mammary tumor formation, we orthotopically transplanted invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once primary tumors were established in recipient mice, we mimicked the clinical course of treatment by conducting a mastectomy. After surgery, recipient mice succumbed to widespread overt metastatic disease in lymph nodes, lungs, and gastrointestinal tract. Genomic profiling of paired mammary tumors and distant metastases showed that our model provides a unique tool to further explore the biology of metastatic disease. Neoadjuvant and adjuvant intervention studies using standard-of-care chemotherapeutics showed the value of this model in determining therapeutic agents that can target early- and late-stage metastatic disease. In obtaining a more accurate preclinical model of metastatic lobular breast cancer, our work offers advances supporting the development of more effective treatment strategies for metastatic disease.

Development of metastatic HER2(+) breast cancer is independent of the adaptive immune system.

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. Copyright

Morphine does not facilitate breast cancer progression in two preclinical mouse models for human invasive lobular and HER2⁺ breast cancer.

Abstract 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.

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1F/F; Trp53F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright

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.

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