Microenvironment In Brain Metastases Research Articles

Tumor Cells and Micro-environment in Brain Metastases

近年来，随着早期诊断的方法的出现及精准治疗的应用，肺癌患者的生存及生活质量都得到很大改善。然而，对于肺癌的脑转移病灶，目前仍缺乏一个理想的治疗方案，严重影响了该部分患者生存状态。了解肿瘤细胞如何在中枢神经系统定植、生长和侵袭等相关生物学行为及其产生机制对预防及治疗肿瘤细胞脑转移病灶具有重大的意义。“种子-土壤”这一假说可以很好的解释这一过程，这一假说的关键即肿瘤细胞可与中枢神经系统微环境各组成之间产生相互适应性变化，正是这种相互作用决定了脑转移病灶的发生发展。本文就脑转移肿瘤细胞、脑转移肿瘤微环境及他们之间的相互作用进行综述，旨在为脑转移病灶的治疗提供新的思路。

Abstract C30: The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression

Abstract Purpose of Study: Research over the past decade has established a critical role for the tumor microenvironment in facilitating tumor growth and promoting invasiveness. Cells of the myeloid lineage, including macrophages, have been known to be key mediators of tumor progression and facilitate metastasis. Recent work from our lab highlights the Ets transcription factors, PU.1 and ETS2, as important players in tumor associated macrophages (TAMs) during breast cancer progression. Both transcription factors are downstream effectors of the Colony Stimulating Factor 1 (CSF1) signaling pathway, which is not only important for myeloid cell survival, but also has a well-established role within the tumor microenvironment. Here, we investigated the requirement of PU.1 in TAMs in a mouse mammary tumor model and have begun to address the role of PU.1 during metastasis. Research Method: We employed cre-loxP technology to conditionally delete PU.1 in the myeloid cell compartment. Syngeneic mice were orthotopically injected with mammary tumor cells directly into the fat pad. To model breast cancer brain metastasis in the mouse, we employed an intracranial injection model which recapitulates the biology of the human disease. We were able to isolate tumor associated macrophages from either site (mammary or brain tumor) for downstream applications. Novel Findings: Macrophage-specific deletion of PU.1 resulted in a significant reduction in mammary tumor growth as well as tumor angiogenesis. Our results suggest that ETS2 and PU.1, acting downstream of the CSF1 signaling pathway, cooperatively regulate the expression of pro-tumor genes along with ‘oncogenic’ microRNA. Conventional ChIP assays show that both transcription factors occupy enhancer regions adjacent to a set of selected genes and cooperatively regulate expression in tumor infiltrating macrophages. We focused on miR-21 and miR-29a for a more detailed investigation as both are expressed in CSF1R+ myeloid cells within the brain metastatic microenvironment. In our intracranial injection model, macrophages (F4/80 positive cells) are recruited to the growing tumor, and both miR-21 and miR-29a are upregulated in TAMs isolated by FACS from tumor-bearing mice as compared to controls. Conclusions and Implications: Given our present data, we believe that the Ets factors, PU.1 and Ets2, regulate a transcriptional program, including microRNAs, that is critical for CSF1 action in tumor infiltrating macrophages. Currently, clinical trials are underway using CSF1R inhibitors for several solid tumor types [clinicaltrials.gov]. The macrophage-specific microRNA, including miR-21 and miR-29a, may serve as indicators for to the efficacy of CSF1R inhibition. Citation Format: Katie Thies, David A. Taffany, Haritha Mathsyaraja, Sudarshana M. Sharma, Walter Hans Meisen, Tom Liu, Cynthia Timmers, Jose Otero, Balveen Kaur, Michael C. Ostrowski. The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C30.

Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC).

Brain metastases (BM) are frequent in small cell lung cancer (SCLC). Novel insights into their pathobiology are needed for development of better therapies. We investigated tumor-infiltrating lymphocyte (TIL) subsets (CD3+, CD8+, CD45RO+, FOXP3+ and PD-1+) and expression of PD-L1 in a series of 32 SCLC BM specimens and four matched primary tumor specimens using immunohistochemistry. 30/32 (93.8 %) BM specimens showed TIL infiltration. CD3+ TILs were observed in 30/32 (93.8 %) BM specimens, CD8+ TILs in 25/32 (78.1 %), CD45RO+ TILs in 15/32 (46.9 %), FOXP3+ TILs in 15/32 (46.9 %) and PD-1+ TILs in 1/32 (3.1 %) BM specimens. Patients with infiltration of CD45RO+ TILS had a significantly longer median survival time (11months; 95 % CI 0.000-26.148) as compared to patients without the presence of CD45RO+ TILs (5months; 95 % CI 0.966-9.034; p = 0.007; log rank test). Membranous PD-L1 on tumor cells was observed in 24/32 (75.0 %) BM specimens, with 11/32 (34.4 %) cases showing PD-L1 expression in over 5 % of viable BM tumor cells. PD-L1 expression on TILs was seen in 8/32 (25.0 %) and on tumor infiltrating macrophages in 9/32 (28.1 %) cases. Patients with PD-L1 expression on TILs presented with improved survival prognosis (6 versus 29months; p = 0.002; log rank test). Among matched primary tumors, all (4/4; 100 %) specimens showed TIL infiltration, while PD-L1 expression found in only 1/4 (25.0 %) specimen. TIL infiltration and PD-L1 expression are commonly found in SCLC BM and presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seem to associate with favorable survival times. Our data suggest an active immune microenvironment in SCLC BM that may be targetable by immune-modulating drugs.

Immune Checkpoint Inhibitors in Brain Metastases: From Biology to Treatment.

Cancer immunotherapy has been a subject of intense research over the last several years, leading to new approaches for modulation of the immune system to treat malignancies. Immune checkpoint inhibitors (anti-CLTA-4 antibodies and anti-PD-1/PD-L1 antibodies) potentiate the host's own antitumor immune response. These immune checkpoint inhibitors have shown impressive clinical efficacy in advanced melanoma, metastatic kidney cancer, and metastatic non-small cell lung cancer (NSCLC)-all malignancies that frequently cause brain metastases. The immune response in the brain is highly regulated, challenging the treatment of brain metastases with immune-modulatory therapies. The immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes in certain patients and, therefore, may serve as a potential treatment target. However, clinical data of the efficacy of immune checkpoint inhibitors in brain metastases compared with extracranial metastases are limited, as most clinical trials with these new agents excluded patients with active brain metastases. In this article, we review the current scientific evidence of brain metastases biology with specific emphasis on inflammatory tumor microenvironment and the evolving state of clinical application of immune checkpoint inhibitors for patients with brain metastases.

Immune Checkpoint Inhibitors in Brain Metastases: From Biology to Treatment

Cancer immunotherapy has been a subject of intense research over the last several years, leading to new approaches for modulation of the immune system to treat malignancies. Immune checkpoint inhibitors (anti–CLTA-4 antibodies and anti–PD-1/PD-L1 antibodies) potentiate the host’s own antitumor immune response. These immune checkpoint inhibitors have shown impressive clinical efficacy in advanced melanoma, metastatic kidney cancer, and metastatic non–small cell lung cancer (NSCLC)—all malignancies that frequently cause brain metastases. The immune response in the brain is highly regulated, challenging the treatment of brain metastases with immune-modulatory therapies. The immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes in certain patients and, therefore, may serve as a potential treatment target. However, clinical data of the efficacy of immune checkpoint inhibitors in brain metastases compared with extracranial metastases are limited, as most clinical trials with these new agents excluded patients with active brain metastases. In this article, we review the current scientific evidence of brain metastases biology with specific emphasis on inflammatory tumor microenvironment and the evolving state of clinical application of immune checkpoint inhibitors for patients with brain metastases.

The inflammatory microenvironment in brain metastases: potential treatment target?

Brain metastases (BM) are frequent in cancer patients and are associated with high morbidity and mortality. The incidence of BM is increasing and only limited treatment strategies are available. Immunomodulatory agents are emerging as effective cancer therapeutics and a deeper understanding of the inflammatory microenvironment in BM might reveal new treatment possibilities. Although the brain has long been considered an 'immune-privileged' organ with limited capacity for inflammatory response, BM do contain tumor infiltrating lymphocytes (TILs). Moreover, dense infiltration with TIL showed an association with favorable patients' overall survival times. Microglia/macrophages were shown to be involved in the pivotal steps of BM formation by providing a "pre-metastatic niche" attracting BM initiating cells and to promote growth and survival of tumor cells in the CNS microenvironment. The anti-cytotoxic T lymphocyte antigen 4 (CTLA4) immune checkpoint inhibitor ipilimumab showed activity against melanoma BM in a clinical trial, thus providing proof of concept for effective therapeutic immunomodulation in patients with CNS metastases. Collectively, the available data show that BM harbors an active inflammatory microenvironment that may be exploited as treatment target.

Abstract 1442: PDGFRβ activation defines a subset of astrocytes in the neuroinflammatory microenvironment of breast cancer brain metastasis, inhibitable by pazopanib.

Abstract Brain metastases occur in approximately 35% of metastatic breast cancer patients whose tumors overexpress HER2. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes and neurons. While a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using a model system to study experimental brain metastasis of breast cancer, we intensively characterized the brain metastatic microenvironment of a brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma subline (231-BR-HER2). The 231-BR-HER2 cell line was injected into the left cardiac ventricle of immunocompromised mice; mice were treated with vehicle, 30mg/kg or 100mg/kg pazopanib twice a day beginning on day 3 post-injection, for 4 weeks. Pazopanib, a multispecific tyrosine kinase inhibitor targeting VEGFR-1, -2, and -3, PDGFR-α and β, and c-kit, directly targeted tumor cells through the disruption of B-Raf pathway, prevents the outgrowth of 231-BR-HER2 large brain metastases by 73% (p<0.0001). Here we evaluated the effect of pazopanib on the brain micro-environment microenvironment. A novel subpopulation of metastasis-associated astrocytes expressing phosphorylated (Y751) PDGFRβ was identified surrounding brain metastases. This subpopulation of p751-PDGFRβ + astrocytes was also identified in human brain metastases from five different craniotomy specimens. Expression of p751-PDGFRβ was studied in primary cultures of astrocyte-enriched glial cells. p751-PDGFRβ expression was increased in response to soluble factors from cultured breast cancer cells, and was inhibited by pazopanib treatment. In the mouse mode, pazopanib treatment resulted in a 70% (p=0.023) decrease of the p751-PDGFRβ+ astrocyte population, at the lowest dose of 30mg/kg, twice daily. In contrast, pazopanib did not have any effect on microglia expression. Collectively, the data identify a new subpopulation of activated astrocytes in the perivascular stage of subclinical brain metastases and demonstrate that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain metastases from breast cancer patients. Citation Format: Brunilde M. Gril, Diane Palmieri, Yong Qian, David J. Liewehr, Seth M. Steinberg, Talha Anwar, Zoraida Andreu, Daniel Masana, Patricia S. Steeg, Fernando Vidal-Vanaclocha. PDGFRβ activation defines a subset of astrocytes in the neuroinflammatory microenvironment of breast cancer brain metastasis, inhibitable by pazopanib. [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 1442. doi:10.1158/1538-7445.AM2013-1442

Pazopanib Inhibits the Activation of PDGFRβ-Expressing Astrocytes in the Brain Metastatic Microenvironment of Breast Cancer Cells

Brain metastases occur in more than one-third of metastatic breast cancer patients whose tumors overexpress HER2 or are triple negative. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes, and neurons. Although a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using an experimental brain metastasis model, we characterized the brain metastatic microenvironment of brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma cells (231-BR-HER2). A previously unidentified subpopulation of metastasis-associated astrocytes expressing phosphorylated platelet-derived growth factor receptor β (at tyrosine 751; p751-PDGFRβ) was identified around perivascular brain micrometastases. p751-PDGFRβ(+) astrocytes were also identified in human brain metastases from eight craniotomy specimens and in primary cultures of astrocyte-enriched glial cells. Previously, we reported that pazopanib, a multispecific tyrosine kinase inhibitor, prevented the outgrowth of 231-BR-HER2 large brain metastases by 73%. Here, we evaluated the effect of pazopanib on the brain neuroinflammatory microenvironment. Pazopanib treatment resulted in 70% (P = 0.023) decrease of the p751-PDGFRβ(+) astrocyte population, at the lowest dose of 30 mg/kg, twice daily. Collectively, the data identify a subpopulation of activated astrocytes in the subclinical perivascular stage of brain metastases and show that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain micrometastases in breast cancer patients.

INVERSE EXPRESSION OF NEUROTROPHINS AND NEUROTROPHIN RECEPTORS AT THE INVASION FRONT OF HUMAN-MELANOMA BRAIN METASTASES

Neurotrophins (NT), such as nerve growth factor (NGF), stimulate the growth and differentiation of several neuronal subpopulations in a distinct yet overlapping manner. Brain-metastatic human melanoma cells overexpress p75(NTR), the low-affinity neurotrophin receptor, and treatment of brain-metastatic cells with NGF stimulates extracellular matrix invasion and production of degradative enzymes in relation to the cellular expression of p75(NTR) Although human melanoma cells express high affinity neurotrophin receptors, such as TrkC (the putative receptor for NT-3), they do not express TrkA, the high-affinity NGF receptor. Using digoxigenin-labeled sense/antisense riboprobes against human p75(NTR) and NGF for in situ hybridization, we determined whether the expression of p75(NTR) and NGF mRNAs are related to brain metastasis of human melanoma. We detected p75(NTR) mRNA at the invasion front of human melanoma brain metastases, whereas p75(NTR) expression was not found in adjacent tissues. In contrast, human NGF mRNA levels were increased in tissues surrounding the melanoma lesions, supporting the notion that NGF and NT are important in determining melanoma brain-metastatic microenvironment. Using antibodies specific to p75(NTR), TrkC, NGF and related NT we found high but heterogeneous levels of p75(NTR) and TrkC expression in malignant melanomas metastatic to the brain. Lower levels of expression were found in primary melanomas or in metastatic melanomas to sites other than brain. Additionally, we found elevated levels of synthesis of NGF and NT-3 but not brain-derived neurotrophic factor (BDNF) or NT-4/5 in the brain tissues surrounding melanoma lesions. These studies support a role for NT and their receptors in the progression of melanomas to the brain-metastatic phenotype.