Brain Metastatic Cells Research Articles

TAMI-06. TUMOR CELL-DERIVED CYTOKINE EXPRESSION CHANGES ASSOCIATED WITH BRAIN METASTASIS IN A SYNGENEIC MOUSE MODEL OF BREAST CANCER

Abstract Breast cancer is the most common malignancy in women in the United States, and brain metastases occur in almost a third of patients with metastatic dissemination. Immunoediting is a critical component of metastatic tumor cell elimination, and tumor clones that develop immune-escape mechanisms are associated with progression and metastatic dissemination. We hypothesized that breast cancer brain metastatic cells harbor immunomodulatory cytokine expression changes that promote an immunosuppressive environment to avoid immune cell-mediated elimination. To study this, a syngeneic mouse model of metastatic breast cancer was used. A brain metastatic line derived from the 4T1 breast cancer parental cell line was created by serially selecting brain metastatic populations of cells after intracardiac injection (4T1 BrM). A gene-expression analysis using an 800-gene cancer immunology-specific microarray panel was performed comparing the 4T1 parental and 4T1 BrM lines. 4T1 BrM cells demonstrate gene expression changes promoting immunosuppression including significant upregulation of IL18 and Lgals9 (Galectin-9) and downregulation of CD40, IL2rg, CCL2, and EOMES. When compared to 4T1 parental lines, the 4T1 BrM line demonstrated decreased expression of CCL2 and increased expression of GM-CSF on a cytokine array, corresponding to results obtained from gene expression analysis. These results suggest tumor-intrinsic cytokine expression changes that may mediate an immunosuppressive environment.

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6–7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.

Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis.

BackgroundPlatinum‐based chemotherapy is effective in inducing shrinkage of primary lung cancer lesions; however, it shows finite therapeutic efficacy in patients suffering from brain metastasis (BM). The intrinsic changes of BM cells, which contribute to the poor results remain unknown.MethodsPlatinum drug‐sensitivity was assessed by utilizing a preclinical BM model of PC9 lung adenocarcinoma cells in vitro and in vivo. High consumption of glutathione (GSH) and two associated upregulated proteins (GPX4 and GSTM1) in BM were identified by integrated metabolomics and proteomics in cell lines and verified by clinical serum sample. Gain‐of‐function and rescue experiments were implemented to reveal the impact and mechanism of GPX4 and GSTM1 on the chemosensitivity in BM. The interaction between GPX4 and GSTM1 was examined by immunoblotting and immunoprecipitation. The mechanism of upregulation of GPX4 was further uncovered by luciferase reporter assay, immunoprecipitation, and electrophoretic mobility shift assay.ResultsThe derivative brain metastatic subpopulations (PC9‐BrMs) of parental cells PC9 developed obvious resistance to platinum. Radically altered profiles of BM metabolism and protein expression compared with primary lung cancer cells were described and GPX4 and GSTM1 were identified as being responsible for the high consumption of GSH, leading to decreased chemosensitivity by negatively regulating ferroptosis. Besides, GSTM1 was found regulated by GPX4, which was transcriptionally activated by the Wnt/NR2F2 signaling axis in BM.ConclusionsCollectively, our findings demonstrated that Wnt/NR2F2/GPX4 promoted acquired chemoresistance by suppressing ferroptosis with high consumption of GSH. GPX4 inhibitor was found to augment the anticancer effect of platinum drugs in lung cancer BM, providing novel strategies for lung cancer patients with BM.

Zinc Favors Triple-Negative Breast Cancer's Microenvironment Modulation and Cell Plasticity.

Triple-negative breast cancer (TNBC) tends to metastasize to the brain, a step that worsens the patient’s prognosis. The specific hallmarks that determine successful metastasis are motility and invasion, microenvironment modulation, plasticity, and colonization. Zinc, an essential trace element, has been shown to be involved in all of these processes. In this work, we focus our attention on the potential role of zinc during TNBC metastasis. We used MDA-MB-BrM2 (BrM2) cells, a brain metastasis model derived from the parental TNBC cell line MDA-MB-231. Our studies show that BrM2 cells had double the zinc content of MDA-MB-231 cells. Moreover, exploring different metastatic hallmarks, we found that the zinc concentration is especially important in the microenvironment modulation of brain metastatic cells, enhancing the expression of SerpinB2. Furthermore, we show that zinc promotes the tumorigenic capacity of breast cancer stem cells. In addition, by causing a disturbance in MDA-MB-231 zinc homeostasis by overexpressing the Zip4 transporter, we were able to increase tumorigenicity. Nevertheless, this strategy did not completely recapitulate the BrM2 metastatic phenotype. Altogether, our work suggests that zinc plays an important role in the transformative steps that tumoral cells take to acquire tumorigenic potential and niche specificity.

BSCI-19. Therapeutic intervention of lung-, breast-, and melanoma-brain metastasis

BackgroundThe incidence of brain metastases (BM) is tenfold higher than that of primary brain tumours. BM predominantly originate from primary lung, breast, and melanoma tumours with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM.MethodsUsing a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft models of BM and captured a “premetastatic” population of BM cells that have just seeded the brains of mice before forming clinically detectable tumours: a cell population that is impossible to detect in human patients but represents a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable tumours.ResultsRNA sequencing of pre-metastatic BM cells from all three primary tumour models with subsequent Connectivity Map analysis identified a lead compound that exhibits selective anti-BM activity in vitro. Preliminary in vivo work has shown that this lead compound reduces the tumor burden of treated mice compared to vehicle control while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BM phenotype.ConclusionTherapeutic targeting of premetastatic BM cells could prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients.

BSCI-07. Multiomics characterization of brain metastases in multiple histologies identifies enrichment of oxidative phosphorylation as a promising therapeutic target

PurposeBrain metastasis (BM) is a lethal complication from systematic malignant tumors, and the incidence is approximately 10–30% of patients with advanced cancer. Extensive genomic analyses with large sample sets and the following functional studies revealed clinically relevant characteristics for BMs. However, these studies have not identified specific abnormalities driving BM in multiple tumor histologies yet. To identify molecular pathogenesis and promising therapeutic targets shared across multiple histologies of BMs, we performed multiomics molecular profiling, along with functional studies using in vitro and in vivo BM models.MethodsFrozen tissues of patient-matched BMs and primary tumors (or extracranial metastases) from breast cancer (N= 14), lung cancer (N = 14) and renal cell carcinomas (N = 7) patients were carried out whole-exome sequencing, mRNA-Seq and reverse-phase protein array. Paired parental and brain metastatic derivatives of MDA-MB-231 and BT474 were examined to assess findings from the multiomics datasets. SCID/beige mice were inoculated with MDA-IBC3 cells via tail vein injection and administered an oxidative phosphorylation (OXPHOS) inhibitor by oral gavage daily for 96 days.ResultsThe multiomics molecular profiling identified enrichment of OXPHOS shared across the histologies of BMs. Brain metastatic derivative cell lines also demonstrated enhanced oxidative metabolism, along with the sensitivity to an OXPHOS inhibitor. Moreover, in vivo studies revealed that OXPHOS inhibition significantly impaired the formation of BM, and fresh brain metastatic derivatives from the murine BM model exhibited the higher oxidative metabolism and sensitivity to the OXPHOS inhibitor as with the prior in vitro studies.ConclusionsOur multiomics characterization of BMs demonstrates heightened oxidative metabolism shared across the multiple histologies, and the OXPHOS inhibition affects more effectively for brain metastatic derivatives rather than the parentals. Further investigation focusing on metabolic abnormalities in BM will likely develop promising therapeutic strategies against BMs.

Abstract 2715: Tamoxifen suppresses brain metastasis of estrogen receptor deficient breast cancer by skewing microglia polarization and enhancing their immune functions

Abstract Brain metastasis of breast cancer exhibits exceedingly poor prognosis, and both triple negative and Her2+ subtypes have the highest incidence of brain metastasis. Although estrogen blockers are considered to be ineffective for their treatment, recent evidence indicates that estrogen blockade using tamoxifen showed certain efficacy. However, how estrogen affects brain metastasis of triple negative breast cancer (TNBC) remains elusive. The communications between neoplastic cells and microglia, especially M2 type pro-tumor microglia, affect brain metastasis progression. Therefore, we hypothesize that estrogen skews microglial polarization to M2 phenotype which promotes brain metastasis. We found that M2 microglia were abundantly infiltrated in brain metastatic lesions of pre-menopausal TNBC patients. To test the effect of estrogen on brain metastasis, we transplanted the SKBrM brain metastatic cell TNBC line into nude mice by intra-cardiac injection followed by with or without 17β-estradiol (E2) treatment. We found that brain metastasis-free survival was decreased, while infiltration of M2 microglia was increased in E2-treated group. Blocking estrogen signaling either by tamoxifen treatment or surgical resection of ovaries suppressed M2 microglial polarization and brain metastasis. To test the effect of E2-driven microglia on tumor progression, brain metastatic cells were treated with conditioned medium (CM) generated from microglia that were treated with or without E2. We found that CM promoted cell growth and increased the population of cancer stem cells. Blocking E2-mediated M2 polarization by tamoxifen suppressed M2 polarization and decreased the secretion of C-C motif chemokine ligand 5, resulting in significant suppression of brain metastasis. To examine the mechanism by which estrogen suppresses anti-tumor function of microglia, we measured the expression of “do not eat me” signal, SIRP on microglia and CD47 on tumor cells. When SKBrM and microglial cells were treated with E2, we found that the protein expression of SIRP and CD47 were up-regulated on microglia and SKBrM, respectively, by E2 treatment. Furthermore, we used flow cytometry to detect the phagocytic ability of microglia by co-culturing SKBrM with microglia. We found that the phagocytic activity of microglia was significantly decreased upon E2 treatment. In contrast, tamoxifen restored E2-suppressed phagocytic ability. In conclusion, our results show that estrogen promotes brain metastasis by skewing polarity of M2 microglia and inhibiting their phagocytic ability, while tamoxifen suppresses brain metastasis by blocking M2 polarization of microglia and increasing their anti-tumor phagocytic ability. Our results also highlight a potential therapeutic utility of tamoxifen for treating brain metastasis of hormone receptor deficient breast cancer. Citation Format: Shih-Ying Wu. Tamoxifen suppresses brain metastasis of estrogen receptor deficient breast cancer by skewing microglia polarization and enhancing their immune functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2715.

Abstract 2647: HOXB7, a key transcriptional regulator in triple negative breast cancer progression

Abstract Despite major advances in breast cancer treatment and management one-third of women with ER-positive, and nearly 30% of women with ER-negative breast cancer will have recurrent disease within fifteen years. Our laboratory and others have accumulated a large body of evidence for the involvement of HOXB7 in various aspects of breast cancer progression: tamoxifen resistance, DNA repair, epithelial-mesenchymal transition, promotion of metastasis in a transgenic mouse model and human cells as well as angiogenesis suggesting a role for HOXB7 not only in the epithelium but also strongly implicating its control on the microenvironment of the tumor. In this project, we focused on understanding factors driven by HOXB7 that lead to triple-negative breast cancer (TNBC). In the previous studies, we found that HOXB7 expression results in the recruitment of stromal components and enhances the formation of lung metastasis in transgenic crosses of HOXB7xHER2 mice. Interestingly, HOXB7 expression was increased in MDA-MB-231 Bone and Brain metastatic cells. Therefore, we hypothesize that HOXB7, through its ability to mediate EMT and recruit pro-angiogenic molecules, is capable of promoting invasion and metastasis. In this report, we established HOXB7 overexpressing MDA-MD-231 cells. We screened secretomes from MB231-HOXB7 cells using Human Cytokine Array Q440 to quantitatively detect 440 human inflammatory factors, growth factors, chemokines, receptors, and cytokines simultaneously. We identified upregulation of IL-8, IL-6, VEGF, ARG2, CXCL1, CF XIV, Procalcitonin, Syndecan-3, AMICA, IL-11, CXCL14, ROBO3, Follistatin, SOST, and ALCAM in the secretomes from MB231-HOXB7 cells. We ranked the expression level of each factor by real-time qRT-PCR and determined that IL-8 and CXCL1 were the top candidates. We confirmed by ELISA that IL-8 and CXCL1 secreted from tumor conditioned medium (TCM) was upregulated compared to the control cells. Our data showed that the HUVEC tube formation was increased by MDA-MD-231-HOXB7 conditioned media. Furthermore, fibroblast and M2 type macrophage migration were promoted by TCM of MDA-MD-231-HOXB7 cells. Significantly, Depletion of IL-8 and CXCL1 using specific shRNAs in MDA-MD-231-HOXB7 cells decreased the HUVEC tube formation and the cell migration of fibroblast and macrophage. In addition, CXCL1 and IL-8 signaling pathway by Reparixin, an inhibitor of the CXCR1/2 abrogated the tube formation and migration induced by MDA-MD-231-HOXB7 cells. These findings implicate IL-8 and CXCL1 signaling as a critical event in HOXB7 overexpression TNBC growth and metastasis via crosstalk between cancer cells and stromal components. Citation Format: Kideok Jin. HOXB7, a key transcriptional regulator in triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2647.

Abstract 7: Modeling cell-cell crosstalk in TNBC brain metastasis

Abstract Brain metastasis (BrM) is a difficult clinical problem in triple-negative breast cancer (TNBC) patients: it occurs in 25% to 40% of metastatic patients while two-year survival rate stubbornly remains a single digit. TNBC usually spreads to the brain rapidly at earlier stages or before the diagnosis of primary cancer. The shorter development time of BrM in TNBC and shorter survival time after BrM diagnosis may indicate a unique innate ability of TNBC tumor cells in adapting to the brain. Indeed, emerging data including ours indicates that TNBC tumor cells have intrinsic brain-like traits resulting in brain-tropism metastasis. Our preliminary studies identified enriched and diverse expressions of receptors and transporters of various neuro-active substances in brain metastatic TNBC cells, including neurotransmitters, gliotransmitters, neurotrophic factors and neural cytokines. By immunopanning astrocytes and microglia/macrophages from mouse brains bearing TNBC brain metastatic tumors at day 3 post tumor cell injection through left cardiac ventricle, and use the CCCExplorer software in modeling the interactions among tumor cells, astrocytes, and microglia/macrophage with cell-type specific RNAseq data, we identified several novel crosstalk signaling, including tumor autocrine TPH1-HTR2, astrocyte→tumor PCDH7-EGFR, IL6-IL6R, CCL5-CCR5 and VGF-C3AR1, and microglia/macrophage→tumor ARTN-GFAR3. Functional experiments demonstrated that cell type-specific depletion of the secreted neuro-active substances deactivated the activities of corresponding receptor and downstream signaling in co-cultured BrM tumor cells. Given that more complex paracrine/autocrine signaling network may dynamically evolve during BrM development, we are characterizing the spatiotemporal cell-cell crosstalk in the BrM metastatic niche that will have important implications for novel therapeutics development. Citation Format: Hong Zhao, Xin Wang, Nikola Liu, Jianting Sheng, Stephen Wong. Modeling cell-cell crosstalk in TNBC brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 7.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.

Paclitaxel (PTX) is a first-line chemotherapeutic drug for breast cancer, but PTX resistance often occurs in metastatic breast cancer. In addition, due to the poor targeting of chemotherapeutic drugs and the presence of the blood-brain barrier (BBB), it is hard to effectively treat brain metastatic breast cancer using paclitaxel. Thus, it is urgent to develop an effective drug delivery system for the treatment of brain metastatic breast cancer. The current study found that TWF1 gene, an epithelial-mesenchymal transition-associated gene, was overexpressed in brain metastatic breast cancer (231-BR) cells and was associated with the PTX resistance of 231-BR cells. Knockdown of TWF1 by small interference RNA (siRNA) in 231-BR cells could effectively increase the sensitivity of brain metastatic breast cancer cells to paclitaxel. Then, a liposome-based drug delivery system was developed for PTX delivery across BBB, enhancing PTX sensitivity and brain metastases targeting via BRBP1 peptide modification. The results showed that BRBP1-modified liposomes could effectively cross the BBB, specifically accumulate in brain metastases, and effectively interfere TWF1 gene expression in vitro and in vivo, and thus they enhanced proliferation inhibition, cell cycle arrest, and apoptosis induction, thereby inhibiting the formation and growth of brain metastases. In summary, our results indicated that BRBP1-modified and PTX- and TWF1 siRNA-loaded liposomes have the potential for the treatment of brain metastatic breast cancer, which lays the foundation for the development of a new targeted drug delivery system.

Delivering Antisense Oligonucleotides across the Blood-Brain Barrier by Tumor Cell-Derived Small Apoptotic Bodies.

The blood‐brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain‐targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain‐targeting drug delivery is explored. It is found that anti‐TNF‐α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO‐loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti‐inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain‐targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale‐up production potential than other EVs.

Brain Metastasizing Breast Cancer Cell Secretome is Modulated by Endoplasmic Reticulum Stress

Introduction Breast cancer is the second leading cause of brain metastasis. 30% of the patients suffering from Triple Negative Breast Cancer (TNBC) develop brain metastasis which confers poor prognosis to breast cancer patients. The mechanisms that facilitate brain metastasis are not fully understood. TNBC cells have high levels of endoplasmic reticulum stress (ER stress) and show activation of the IRE1 pathway of the Unfolded Protein Response (UPR). We showed that MDA-231/BR cells, the brain metastatic subline of MDA-MB-231, have high endogenous levels of IRE1 activity suggesting this UPR pathway to support survival under ER stress. Our research investigates the role of the UPR IRE1 arm in formation of TNBC brain metastasis. Here, we present the effect of ER stress induction and IRE1 inhibition on PERK activation and cytokine secretion in MDA-231/BR. Methods We used the brain metastatic triple-negative cell model MDA-231/BR to study the influence of IRE1 inhibition on other UPR pathways. ER stress was induced by serum-starvation or pharmacologically with Thapsigargin (Tg). MKC8866, a highly selective non-competitive inhibitor of IRE1 RNAse and splicing activity, was used at 10 μM. Successful inhibition IRE1 RNAse activity was assessed by Western blot detection of the spliced XBP1 protein (sXBP1). Modulation of the PERK activity upon ER stress induction and MKC8866 inhibition was determined by Western blot and with caspase-3/7 apoptosis assays. Cell viability and cell migration was assessed by real time cell analysis. Cytokines secreted form MDA-231/BR were determined. Brain endothelial cells were subjected to MDA-231/BR conditioned media and cell viability assays. Gene expression changes of tight junctional proteins claudin-3, claudin-5 and occludin were quantified by qPCR. Results MDA-231/BR cells show constitutive activation of the IRE1 RNAse arm which can be further induced upon serum starvation as well as low concentrations of Thapsigargin (Tg). The IRE1 RNAse inhibitor MKC8866 (10 μM) completely represses sXBP1 under basal and induced ER stress. However, the stressors show different downstream effects: unlike serum starvation, only Tg treatment activates the pro-apoptotic PERK pathway. This is confirmed by reduced cell viability and increased apoptosis upon Tg treatment. Cytokine analysis from culture supernatants of ER stress-induced MDA-231/BR cells showed increased secretion of pro-metastatic cytokines, some were independent of IRE1 activity. Brain endothelial cells exposed to MDA-231/BR conditioned media did not show altered cell viability nor changes in tight-junctional protein expression suggesting direct cell contacts to influence endothelial phenotypes. Conclusions This study showed that moderate ER stress induction by serum starvation, in contrast to pharmacological ER stress induction, does not induce pro-apoptotic UPR activity in MDA-231/BR cells. Increased MDA-231/BR secreted cytokines are not affecting brain endothelial cell viability, proliferation or expression of tight junction proteins.

Prognostic Factors and Current Treatment Strategies for Renal Cell Carcinoma Metastatic to the Brain: An Overview.

Simple SummaryBrain metastases are a commonly recognized poor prognostic factor in patients with cancer. Due to their poor prognosis, these patients were commonly excluded from the most important clinical trials that have revolutionized the oncological clinical practice. Renal cell carcinoma represents one of the most frequent neoplasia that metastasize to the brain. Due to the therapeutical advances the overall survival of brain metastatic renal cell carcinoma is improved even in the absence of tailored studies that are need to plan an adequate therapeutic strategy for these patients.Renal cell carcinoma (RCC) is one of primary cancers that frequently metastasize to the brain. Brain metastasis derived from RCC has the propensity of intratumoral hemorrhage and relatively massive surrounding edema. Moreover, it confers a grim prognosis in a great percentage of cases with a median overall survical (mOS) around 10 months. The well-recognized prognostic factors for brain metastatic renal cell carcinoma (BMRCC) are Karnofsky Performance Status (KPS), the number of brain metastasis (BM), the presence of a sarcomatoid component and the presence of extracranial metastasis. Therapeutic strategies are multimodal and include surgical resection, radiotherapy, such as stereotactic radiosurgery due to the radioresistance of RCC and systemic strategies with tyrosin kinase inhibitors (TKI) or Immune checkpoint inhibitors (ICI) whose efficacy is not well-established in this setting of patients due to their exclusion from most clinical trials. To date, in case of positive prognostic factors and after performing local radical therapies, such as complete resection of BM or stereotactic radiosurgery (SRS), the outcome of these patients significantly improves, up to 33 months in some patients. As a consequence, tailored clinical trials designed for BMRCC are needed to define the correct treatment strategy even in this poor prognostic subgroup of patients.

Tamoxifen suppresses brain metastasis of estrogen receptor-deficient breast cancer by skewing microglia polarization and enhancing their immune functions

BackgroundBrain metastasis of breast cancer exhibits exceedingly poor prognosis, and both triple negative (TN) and Her2+ subtypes have the highest incidence of brain metastasis. Although estrogen blockers are considered to be ineffective for their treatment, recent evidence indicates that estrogen blockade using tamoxifen showed certain efficacy. However, how estrogen affects brain metastasis of triple negative breast cancer (TNBC) remains elusive.MethodsTo examine the effect of estrogen on brain metastasis progression, nude mice were implanted with brain metastatic cells and treated with either estrogen supplement, tamoxifen, or ovariectomy for estrogen depletion. For clinical validation study, brain metastasis specimens from pre- and post-menopause breast cancer patients were examined for microglia polarization by immunohistochemistry. To examine the estrogen-induced M2 microglia polarization, microglia cells were treated with estrogen, and the M1/M2 microglia polarization was detected by qRT-PCR and FACS. The estrogen receptor-deficient brain metastatic cells, SkBrM and 231BrM, were treated with conditioned medium (CM) derived from microglia that were treated with estrogen in the presence or absence of tamoxifen. The effect of microglia-derived CM on tumor cells was examined by colony formation assay and sphere forming ability.ResultsWe found that M2 microglia were abundantly infiltrated in brain metastasis of pre-menopausal breast cancer patients. A similar observation was made in vivo, when we treated mice systemically with estrogen. Blocking of estrogen signaling either by tamoxifen treatment or surgical resection of mice ovaries suppressed M2 microglial polarization and decreased the secretion of C-C motif chemokine ligand 5, resulting in suppression of brain metastasis. The estrogen modulation also suppressed stemness in TNBC cells in vitro. Importantly, estrogen enhanced the expression of signal regulatory protein α on microglia and restricted their phagocytic ability.ConclusionsOur results indicate that estrogen promotes brain metastasis by skewing polarity of M2 microglia and inhibiting their phagocytic ability, while tamoxifen suppresses brain metastasis by blocking the M2 polarization of microglia and increasing their anti-tumor phagocytic ability. Our results also highlight a potential therapeutic utility of tamoxifen for treating brain metastasis of hormone receptor-deficient breast cancer.

Irradiation-Induced Activated Microglia Affect Brain Metastatic Colonization of NSCLC Cells via miR-9/CDH1 Axis.

Background and PurposeBrain metastasis is among the leading causes of death in patients with non-small-cell lung cancer (NSCLC). Through yet unknown mechanisms, prophylactic cranial irradiation (PCI) can significantly decrease the incidence of brain metastases. Given that PCI probably exerts indirect anti-tumoral effects by turning cerebral “soil” unfavorable for the colonization of metastatic tumor “seeds”. This study aims to reveal how PCI regulates the brain microenvironment conducing to a reduction in brain metastases.Materials and MethodsKey markers of M1/M2 microglia types and mesenchymal-to-epithelial transition (MET) were analyzed by qRT-PCR and Western Blot in vitro. The target miR-9 was obtained by miRNA array analysis and confirmed by qRT-PCR in microglia. We used miRTarBase and TargetScan to analyze the target genes of miR-9 and confirmed by luciferase activity assay. Anti-metastatic effects of irradiation on the brain were evaluated by intravital imaging using a brain metastatic A549-F3 cell line in a nude mouse model.ResultsIrradiation induced M1 microglia activation, which inhibited the MET process of A549 cell lines. Furthermore, levels of miR-9 secreted by irradiated M1 microglia significantly increased and played a vital role in the inhibition of the A549 MET process by directly targeting CDH1, concurrently decreasing cell capacity for localization in the brain, thus reducing brain metastases.ConclusionWe demonstrated that miR-9 secreted by irradiated M1-type microglia played an important role in modulating A549 cell lines into mesenchymal phenotype and further decreased their localization capabilities in the brain. Our findings signify the modulating effect of irradiation on metastatic soil and the cross-talk between tumour cells and the metastatic microenvironment; importantly, they provide new opportunities for effective anti-metastasis therapies, especially for brain metastasis patients.

Outcomes and prognostic factors in metastatic renal cell carcinoma patients with brain metastases.

289 Background: The study aimed to evaluate the outcomes and prognostic factors in patients with brain metastatic renal cell carcinoma (bmRCC). Methods: The data of 322 patients with renal cell carcinoma, between 2012 and 2020, were retrospectively reviewed. The clinicopathological features and treatments of the patients with bmRCC were recorded. Overall survival (OS) and prognostic factors were evaluated with Kaplan-Meier analysis and Cox-regression analysis. Results: Forty (12.4%) of the patients had bmRCC. The median follow-up period was 7.3 months (range, 0.2-55.5). The male/female ratio was 2.3, and the median age at diagnosis was 62 years (range, 25-84). Seventeen (42.5%) of the patients were de-novo metastatic, and nine (22.5%) of the patients had brain metastases at presentation. The most common extracranial metastatic sites of the disease were lung (72.5%), bone (47.5%), lymph node (27.5%), and liver (12.5%). Twenty-four (60%) patients previously had received various therapies (tyrosine kinase inhibitor, checkpoint inhibitors, or palliative radiotherapy). After brain metastases developed, 92% of the patients received brain radiotherapy (whole-brain radiotherapy or stereotactic radiosurgery), and twenty-five (62.5%) patients received different therapies. Nine patient received sunitinib, nine patient pazopanib, five patient nivolumab, and two patient axitinib. A total of 32 (80%) patients died during the study period. The median OS was 8.8 months (range, 2.9-14.6) for all patients with bmRCC. Six months- and one-years overall survival ratios were 60% and 40%, respectively. In univariate analysis, the number of brain metastasis (p = 0.352), the localization of brain metastasis (p = 0.790), the longest size of brain metastasis (p = 0.454), the number of extracranial metastatic sites (p = 0.812), de-novo metastatic disease (p = 0.177), primary tumor localization (left or right) (p = 0.903), and tumor grade (p = 0.093) were not statistically significant factors on OS. However, age (p = 0.02), a history of nephrectomy (p < 0.001), receiving brain radiotherapy (p = 0.005), and type of treatment (p = 0.044) was statistically significant. Only, the effect of brain radiotherapy on OS (p = 0.011) was confirmed in multivariate analysis. Conclusions: The prognostic data of patients with bmRCC is limited. In this study, we observed that the prognosis of patients with bmRCC was poor. Despite a small number of patients, we detected that the effect of tyrosine kinase inhibitors and nivolumab was comparable, and receiving brain radiotherapy was a prognostic factor for OS.

Gain of HIF1 Activity and Loss of miRNA let-7d Promote Breast Cancer Metastasis to the Brain via the PDGF/PDGFR Axis.

Early detection and adjuvant therapies have significantly improved survival of patients with breast cancer over the past three decades. In contrast, management of metastatic disease remains unresolved. Brain metastasis is a late complication frequently observed among patients with metastatic breast cancer, whose poor prognosis calls for novel and more effective therapies. Here, we report that active hypoxia inducible factor-1 (HIF1) signaling and loss of the miRNA let-7d concur to promote brain metastasis in a recently established model of spontaneous breast cancer metastasis from the primary site to the brain (4T1-BM2), and additionally in murine and human experimental models of breast cancer brain metastasis (D2A1-BM2 and MDA231-BrM2). Active HIF1 and let-7d loss upregulated expression of platelet-derived growth factor (PDGF) B/A in murine and human brain metastatic cells, respectively, while either individual silencing of HIF1α and PDGF-A/B or let-7d overexpression suppressed brain metastasis formation in the tested models. Let-7d silencing upregulated HIF1α expression and HIF1 activity, indicating a regulatory hierarchy of the system. The clinical relevance of the identified targets was supported by human gene expression data analyses. Treatment of mice with nilotinib, a kinase inhibitor impinging on PDGF receptor (PDGFR) signaling, prevented formation of spontaneous brain metastases in the 4T1-BM2 model and reduced growth of established brain metastases in mouse and human models. These results identify active HIF1 signaling and let-7d loss as coordinated events promoting breast cancer brain metastasis through increased expression of PDGF-A/B. Moreover, they identify PDGFR inhibition as a potentially actionable therapeutic strategy for patients with brain metastatis. SIGNIFICANCE: These findings show that loss of miRNA let-7d and active HIF1 signaling promotes breast cancer brain metastasis via PDGF and that pharmacologic inhibition of PDGFR suppresses brain metastasis, suggesting novel therapeutic opportunities. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/594/F1.large.jpg.See related article by Thies et al., p. 606.

Development and characterization of patient-derived xenografts from non-small cell lung cancer brain metastases

Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

Circular RNA circBCBM1 promotes breast cancer brain metastasis by modulating miR-125a/BRD4 axis.

Circular RNAs (circRNAs) play critical roles in tumorigenesis and the progression of various cancers. We previously identified a novel upregulated circRNA, circBCBM1 (hsa_circ_0001944), in the context of breast cancer brain metastasis. However, the potential biological function and molecular mechanism of circBCBM1 in breast cancer brain metastasis remain largely unknown. In this study, we confirmed that circBCBM1 was a stable and cytoplasmic circRNA. Functionally, circBCBM1 promoted the proliferation and migration of 231-BR cells in vitro and growth and brain metastasis in vivo. Mechanistically, circBCBM1 acted as an endogenous miR-125a sponge to inhibit miR-125a activity, resulting in the upregulation of BRD4 (bromodomain containing 4) and subsequent upregulation of MMP9 (matrix metallopeptidase 9) through Sonic hedgehog (SHH) signaling pathway. Importantly, circBCBM1 was markedly upregulated in the breast cancer brain metastasis cells and clinical tissue and plasma samples; besides, circBCBM1 overexpression in primary cancerous tissues was associated with shorter brain metastasis-free survival (BMFS) of breast cancer patients. These findings indicate that circBCBM1 is involved in breast cancer brain metastasis via circBCBM1/miR-125a/BRD4 axis. CircBCBM1 may serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for breast cancer brain metastasis.