Mouse Model Of Bone Metastasis Research Articles

Phosphate Ion-Responsive and Calcium Peroxide-Based Nanomedicine for Bone-Targeted Treatment of Breast Cancer Bone Metastasis.

The treatment of breast cancer bone metastasis is an unresolved clinical challenge, mostly because currently therapeutic approaches cannot simultaneously block the tumor growth and repair the osteolytic bone injuries at the metastatic site. Herein, the study develops a novel nanomedicine to treat breast cancer bone metastasis. The nanomedicine is based on phosphate ion-responsive and calcium peroxide-based nanoparticles carrying the bone-targeting agent zoledronic acid on the surface and loaded with the photosensitizer indocyanine green. Following intravenous administration to a mouse model of breast cancer bone metastasis, the nanoparticles efficiently accumulate at the bone metastasis site, react with free phosphate ions, and form hydroxyapatite nanoaggregates and O2, while releasing the photosensitizer. Hydroxyapatite nanoaggregates elicit the remineralization of the collagenous bone matrix and trigger tumor cell apoptosis. Upon irradiating tumor-bearing legs with an 808nm laser source, the O2 and free photosensitizer produced 1O2 by the reaction of the nanoparticles with phosphate ions, further boosting the anti-tumor effect. Tumor killing hampers the vicious cycle at the site of bone metastasis, translating to osteolysis blockade and further encouraging the remineralization of bone matrix. This work sheds light on the development of a novel, safe, and efficient approach for the treatment of breast cancer bone metastasis.

Cryoprotective isoliquiritigenin-zein phosphatidylcholine nanoparticles inhibits breast cancer-bone metastasis by targeting JAK-STAT signaling pathways

Breast cancer (BC) is the most common cancer in women and is known for its tendency to spread to the bones, causing significant health issues and mortality. In this study, we aimed to investigate whether cryoprotective isoliquiritigenin-zein phosphatidylcholine nanoparticles (ISL@ZLH NPs) could inhibit BC-induced bone destruction and tumor metastasis in both in vitro and animal models. To evaluate the potential of ISL@ZLH NPs, we conducted various experiments. First, we assessed cell viability, colony formation, transwell migration, and wound healing assays to determine the impact of ISL@ZLH NPs on BC cell behavior. Western blotting, TRAP staining and ALP activity were performed to examine the effects of ISL@ZLH NPs on osteoclast formation induced by MDA-MB-231 cell-conditioned medium and RANKL treated RAW 264.7 cells. Furthermore, we assessed the therapeutic impact of ISL@ZLH NPs on tumor-induced bone destruction using a mouse model of BC bone metastasis. Treatment with ISL@ZLH NPs effectively suppressed BC cell proliferation, colony formation, and motility, reducing their ability to metastasize. ISL@ZLH NPs significantly inhibited osteoclast formation and the expression of factors associated with bone destruction in BC cells. Additionally, ISL@ZLH NPs suppressed JAK-STAT signaling in RAW264.7 cells. In the BCBM mouse model, ISL@ZLH NPs led to a significant reduction in osteolytic bone lesions compared to the control group. Histological analysis and TRAP staining confirmed that ISL@ZLH NPs preserved the integrity of bone structure, preventing invasive metastasis by confining tumor growth to the bone marrow cavity. Furthermore, ISL@ZLH NPs effectively suppressed tumor-induced osteoclastogenesis, a key process in BC-related bone destruction. Our findings demonstrate that ISL@ZLH NPs have the potential to inhibit BC-induced bone destruction and tumor metastasis by targeting JAK-STAT signaling pathways and suppressing tumor-induced osteoclastogenesis. These results underscore the therapeutic promise of ISL@ZLH NPs in managing BC metastasis to the bones.

Abstract 2752: ICA1 promotes prostate cancer progression and metastasis via AR down-regulation

Abstract Cancer metastasis, one of the main characteristics in malignancy, contributes to high mortality in cancer patients. Prostate cancer (PCa) is the most common cancer among men and the second leading cause of cancer-related death in Europe and the United States. In China, its incidence has been dramatically increased over past years. However, the mechanism of PCa progression and metastasis remains unclear. The androgen receptor (AR) has been demonstrated to play an important role in PCa progression and metastasis. We have previously established mouse models of PCa bone metastasis and PCa lung metastasis. We have obtained cell clones with bone-specific or lung-specific metastatic capability. By transcriptomic analysis of highly metastatic cell clones and their parental cells, we found a gene group including islet cell autoantigen 1 (ICA1) that are highly expressed in both metastatic cell clones. We further compared these results with RNA array data in clinical samples. Data comparison and correlation analysis showed that ICA1 was negatively correlated with AR expression. Survival analysis of PCa and lung cancer patients showed higher expression of ICA1 but with shorter overall survival. Down-regulation of ICA1 expression in highly metastatic tumor cells significantly inhibited cell growth, colony formation, migration, and invasion. ICA1 overexpression in parental RM1 cells apparently increased cell growth, migration, and metastasis. Interestingly, down-regulation of ICA1 also significantly activated AMPK/mTOR signaling pathway. These findings are anticipated to shed light on the novel pathogenic mechanism underlying PCa and propose a compelling theoretical basis for the application of ICA1 in PCa. This work supported by the NSFC projects (81972420, 81972766, 82173336), as well as grants from the Science and Technology Project of Shenzhen (JCYJ20210324104214040, JCYJ20190809161811237).Keywords: prostate cancer, metastasis, ICA1, androgen receptor Citation Format: Fuhao Wang, Yu He, Xin Huang, Ming Chang, Xiao Zhang, Xiaoyi Li, Xinyu Ye, Yi Lu. ICA1 promotes prostate cancer progression and metastasis via AR down-regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2752.

Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

Combining TIGIT Blockade with MDSC Inhibition Hinders Breast Cancer Bone Metastasis by Activating Antitumor Immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis is incurable and is associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that cross-talk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the proinflammatory cytokine IL1β as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating antitumor immunity. Analysis of patient samples revealed that TIGIT and IL1β are prominent in human bone metastasis. Our findings suggest that cotargeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis. Significance: Temporal transcriptome profiling of the immune microenvironment in breast cancer bone metastasis revealed key communication pathways between dysfunctional T cells and myeloid derived suppressor cells. Cotargeting of TIGIT and IL1β inhibited bone metastasis and improved survival. Validation in patient data implicated these targets as a novel promising approach to treat human bone metastasis.

From breast cancer cell homing to the onset of early bone metastasis: The role of bone (re)modeling in early lesion formation.

Breast cancer often metastasizes to bone, causing osteolytic lesions. Structural and biophysical changes are rarely studied yet are hypothesized to influence metastasis. We developed a mouse model of early bone metastasis and multimodal imaging to quantify cancer cell homing, bone (re)modeling, and onset of metastasis. Using tissue clearing and three-dimensional (3D) light sheet fluorescence microscopy, we located enhanced green fluorescent protein-positive cancer cells and small clusters in intact bones and quantified their size and spatial distribution. We detected early bone lesions using in vivo microcomputed tomography (microCT)-based time-lapse morphometry and revealed altered bone (re)modeling in the absence of detectable lesions. With a new microCT image analysis tool, we tracked the growth of early lesions over time. We showed that cancer cells home in all bone compartments, while osteolytic lesions are only detected in the metaphysis, a region of high (re)modeling. Our study suggests that higher rates of (re)modeling act as a driver of lesion formation during early metastasis.

Unique macrophage phenotypes activated by BMP signaling in breast cancer bone metastases.

Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow-derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.

Development of a New Focal Mouse Model of Bone Metastasis in Renal Cell Carcinoma.

Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Inhibiting Osteolytic Breast Cancer Bone Metastasis by Bone-Targeted Nanoagent via Remodeling the Bone Tumor Microenvironment Combined with NIR-II Photothermal Therapy.

Bone is one of the prone metastatic sites of patients with advanced breast cancer. The "vicious cycle" between osteoclasts and breast cancer cells plays an essential role in osteolytic bone metastasis from breast cancer. In order to inhibit bone metastasis from breast cancer, NIR-II photoresponsive bone-targeting nanosystems (CuP@PPy-ZOL NPs) are designed and synthesized. CuP@PPy-ZOL NPs can trigger the photothermal-enhanced Fenton response and photodynamic effect to enhance the photothermal treatment (PTT) effect and thus achieve synergistic anti-tumor effect. Meanwhile, they exhibit a photothermal enhanced ability to inhibit osteoclast differentiation and promote osteoblast differentiation, which reshaped the bone microenvironment. CuP@PPy-ZOL NPs effectively inhibited the proliferation of tumor cells and bone resorption in the in vitro 3D bone metastases model of breast cancer. In a mouse model of breast cancer bone metastasis, CuP@PPy-ZOL NPs combined with PTT with NIR-II significantly inhibited the tumor growth of breast cancer bone metastases and osteolysis while promoting bone repair to achieve the reversal of osteolytic breast cancer bone metastases. Furthermore, the potential biological mechanisms of synergistic treatment are identified by conditioned culture experiments and mRNA transcriptome analysis. The design of this nanosystem provides a promising strategy for treating osteolytic bone metastases.

Synergistic effect of docetaxel and gambogic acid on bone metastasis of lung cancer

Gambogic acid (GA) as an active compound isolated from Gamboge, have been investigated for many years and proved to be a promising natural anticancer agent for clinical treatment. This study aimed to investigate the inhibitory effect of docetaxel (DTX) combined with gambogic acid on bone metastasis of lung cancer. The anti-proliferation effect of the combination of DTX and GA on Lewis lung cancer (LLC) cells was determined by MTT assays. The anticancer effect of the combination of DTX and GA on bone metastasis of lung cancer in vivo was explored. Evaluation of the efficacy of drug therapy was performed by comparing the degree of bone destruction and the pathological section of bone tissue of the treated mice with that of the control mice. In vitro cytotoxicity, cell migration, and osteoclast-induced formation assay showed that GA enhanced the therapeutic effect of DTX in Lewis lung cancer cell with a synergistic effect. In an orthotopic mouse model of bone metastasis, the average survival of the DTX+GA combination group (32.61d±1.06 d) was significantly increased compared with that of the DTX group (25.75 d±0.67 d) or GA group (23.99 d±0.58 d), *P<0.01. The combination of DTX and GA has synergistic effect and resulted in more effective inhibition of tumor metastasis, providing a strong preclinical rationale for the clinical development of the DTX+GA combination for treating bone metastasis of lung cancer.

Protein Kinase D2 and D3 Promote Prostate Cancer Cell Bone Metastasis by Positively Regulating Runx2 in a MEK/ERK1/2–Dependent Manner

Advanced-stage prostate tumors metastasize to the bone, often causing death. The protein kinase D (PKD) family has been implicated in prostate cancer development; however, its role in prostate cancer metastasis remains elusive. This study examined the contribution of PKD, particularly PKD2 and PKD3 (PKD2/3), to the metastatic potential of prostate cancer cells and the effect of PKD inhibition on prostate cancer bone metastasis invivo. Depletion of PKD2/3 by siRNAs or inhibition by the PKD inhibitor CRT0066101 in AR-positive and AR-negative castration-resistant prostate cancer cells potently inhibited colony formation and cell migration. Depletion or inhibition of PKD2/3 significantly blocked tumor cell invasion and suppressed the expression of genes related to bone metastasis in the highly invasive PC3-ML cells. The reduced invasive activity resulting from PKD2/3 depletion was in part mediated by the transcription factor Runx2, as its silencing decreased PKD2/3-mediated metastatic gene expression through the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 signaling axis. Furthermore, inhibition of PKD by CRT0066101 potently decreased the frequency of bone micrometastases in a mouse model of bone metastasis based on intracardiac injection of PC3-ML cells. These results indicate that PKD2/3 plays an important role in the bone metastasis of prostate cancer cells, and its inhibition may be beneficial for the treatment of advanced prostate cancer.

Mouse Models of Tumor Bone Metastasis and Invasion for Studying CCN Proteins.

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Abstract 2512: The metastatic breast cancer microenvironment in bone exhibits unique BMP signaling

Abstract In 2021, an estimated 281,550 women will be diagnosed with breast cancer (BC) in the U.S., and 43,600 BC patients will succumb to the disease. BC patient prognosis has improved for localized BC, yet metastatic BC continues to cause high mortality with a 5 year survival rate of only 27%. Approximately 70% of BC metastases occur in the bone, with a tumor microenvironment (TME) composed primarily of BC cells, immune cells, and bone cells. In these cellular compartments, bone morphogenetic protein (BMP) signaling exhibits unique TME dependent tumor promoter and suppressor effects. Previous studies in our lab have found BMPs promote myeloid progenitors, polarization of M2 macrophages, and tumor progression in a BMPR1a LysMCre conditional knockout mouse model. Yet to establish BMPs as a viable target for the treatment of metastatic bone, the mechanisms behind BMPs promoting BC bone metastases must be investigated. To further investigate BMP dependent myeloid heterogeneity in cancer, we have utilized a cohort of non-treatment naïve BC patient bone biopsies to unveil the distinct myeloid populations in the TME of BC bone metastases. Differential gene expression analysis revealed a subset of patient samples with a high myeloid gene signature, corresponding with increased chemokine, cytokine and JAK/STAT signaling gene pathways in addition to enhanced BMP signaling. Digital Spatial Profiling via the NanoString GeoMx platform and multiplexed immunohistochemistry staining via the AKOYA Vectra Polaris platform were used to analyze the spatial context of the TME in our cohort of patient bone. We found enhanced myeloid cell infiltration, myeloid heterogeneity and M2 macrophage polarization in the TME of high myeloid gene signature patient samples. This precision oncology analysis into the unique landscape of the metastatic bone TME revealed BMP driven phenotypes in distinct TME cellular components. To then determine if the TME features observed in the metastatic bone samples was reflective of altered innate trained immunity regulated by BMPs, we investigated the requirement for BMP signaling in myeloid inflammatory responses both in vitro and in a mouse model of BC bone metastasis. We found BMPs alter the ability for macrophages to undergo innate trained immunity functions and BC bone metastases alter myeloid inflammatory responses in mice. Investigating the heterogeneity and functions of myeloid cells in the TME of bone metastatic BC will help advance therapeutic target development for BC patients with bone lesions. Expanding therapeutic options for metastatic BC patients will improve patient quality of life and reduce deaths caused by metastasis. Citation Format: Claire Louise Ihle, Desiree Straign, Philip Owens. The metastatic breast cancer microenvironment in bone exhibits unique BMP signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2512.

Abstract 6010: Mouse hind limb tumor metastatic model to evaluate prophylaxis and treatment

Abstract We have established and optimized a robust tumor bone metastasis mouse model, which recapitulates the tumor metastasis progression from circulation to bone colonization, and provides windows for both prophylaxis and treatment. Specifically, we inoculated two breast cancer cell lines, JIMT-1 and MDA-MB-231, each with high metastatic potential, into mouse intra-caudal artery. By detection of steadily incremental bioluminescence signal, prophylaxis was observed to precede metastases, which were found exclusively on mouse hind limbs. These mouse models exhibited swollen hind limbs and even paralysis at late stage. Gross necropsy and pathology analysis confirmed the exclusive hind limb localization, the invasion and spreading of tumor cells to muscles and bone marrows, and the lesions in tibias and femurs. Our robust bone metastases models thus imitate the multiple stages of tumor metastasis, leading to exclusion bone metastasis and opening up windows for prophylaxis and treatment. Citation Format: Yao Shuai, Chaomeng Wang, Ling Tong, Qiao Liu, Jie Xu, Qunsheng Ji. Mouse hind limb tumor metastatic model to evaluate prophylaxis and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6010.

Abstract 3948: Development of bone-targeted Ron inhibitors to treat osseous metastases from breast cancers

Abstract Purpose: About 500,000 people each year worldwide die from metastatic breast cancer. More than 70% of these metastatic breast cancer patients have bone metastases and median survival time from diagnosis of bone metastasis is only 2-3 years. As breast cancer bone metastases are primarily osteolytic, patients suffer from significant morbidities, including severe pain, pathological fractures, nerve compression, and hypercalcemia. Previously, it was shown that RON receptor tyrosine kinase mediates breast cancer outgrowth at metastatic sites, promotes osteolytic bone destruction, and induces systemic immune suppression. We hypothesized that inhibition of RON in specialized resident macrophages and/or osteoclasts will eliminate metastasis through dual effects of blocking osteolysis and enhancing anti-tumor immunity. In this study, we have developed several bis(phosphonate) or phosphate conjugates of BMS-777607 and LY2801653, and report their accumulation in the bone microenvironment along with effects on osteolysis and tumor burden. Methods: We have utilized novel synthetic schemes for synthesis of the drug conjugates. In vitro kinase assays were performed using ADP-Glo RON kinase assay kit (Promega). Hydroxyapatite binding assay was used to determine binding of drug conjugates to the bone mineral. Changes in phosphorylation of RON was demonstrated by Western blotting. Plasma pharmacokinetics and bone accumulation of lead drug conjugates in mice was assessed by mass-spectrometry. An intratibial bone metastasis model in which MSP-expressing MMTV-PyMT tumor cells are injected into the tibia of mice was used to assess the effect of treatment with lead drug conjugates. Results: Seven novel drug conjugates were synthesized and inhibited RON kinase activity with an IC50 of 0.060-2.4 µM. Three drug conjugates demonstrated better hydroxyapatite binding when compared with BMS-777607. In agreement with the in vitro kinase activity, a variable decrease in the phosphorylation of RON was observed in cells treated with the drug conjugates. ZB-28, a monophosphonate derivative of BMS-777607, showed higher accumulation in the bone when compared with BMS-777607 but failed to block tumor-mediated bone osteolysis in an intratibial bone metastasis mouse model. Currently, a cleavable phosphate conjugate of BMS-777607 (ZB-32) is undergoing testing in the same mouse model. We have confirmed that ZB-32 and its hydrolyzed product ZB-33 accumulate in the bone to a significantly higher level than BMS-777607. These results along with preliminary ADME will be presented. Conclusion: Targeting RON kinase in the bone microenvironment is a promising approach to alleviate morbidities observed in breast cancer patients with bone metastases. We present novel bone-targeting conjugates of BMS-777607 and highlight the potential for clinical development. Citation Format: Trason Thode, Jaime Fornetti, Ryan Rodriguez del Villar, Alexis Weston, Serina Ng, Srikanta Dana, Raffaella Soldi, Mohan Kaadige, Hariprasad Vankayalapati, Srinivas Kasibhatla, Alana Welm, Sunil Sharma. Development of bone-targeted Ron inhibitors to treat osseous metastases from breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3948.

NKX2-8/PTHrP Axis-Mediated Osteoclastogenesis and Bone Metastasis in Breast Cancer.

Bone metastasis is one of the most common distant metastasis of breast cancer, which could cause serious skeletal disease and increased cancer-related death. Therefore, identification of novel target(s) to develop therapeutics would improve patient outcomes. The role of NKX2-8 in modulation of bone remodeling was determined using osteoclastogenesis and micro-CT assays. The expression of NKX2-8 was examined via immunohistochemistry analysis in 344 breast cancer tissues. The mechanism underlying NKX2-8-mediated PTHrP downregulation was investigated using biotinylated deactivated Cas9 capture analysis, chromatin immunoprecipitation, co-immunoprecipitation assays. A bone-metastatic mouse model was used to examine the effect of NKX2-8 dysregulation on breast cancer bone metastasis and the impact of three PTHrP inhibitor on prevention of breast cancer bone metastasis. The downregulated expression of NKX2-8 was significantly correlated with breast cancer bone metastasis. In vivo bone-metastatic mouse model indicated that silencing NKX2-8 promoted, but overexpressing NKX2-8 inhibited, breast cancer osteolytic bone metastasis and osteoclastogenesis. Mechanistically, NKX2-8 directly interacted with HDAC1 on the PTHrP promoter, which resulted in a reduction of histone H3K27 acetylation, consequently transcriptionally downregulated PTHrP expression in breast cancer cells. Furthermore, targeting PTHrP effectively inhibited NKX2-8-downregulation-mediated breast cancer bone metastasis. Taken together, our results uncover a novel mechanism underlying NKX2-8 downregulation-mediated breast cancer bone metastasis and represent that the targeting PTHrP might be a tailored treatment for NKX2-8 silencing-induced breast cancer bone metastasis.

Single-cell sequencing reveals MYC targeting gene MAD2L1 is associated with prostate cancer bone metastasis tumor dormancy

BackgroundAmong malignant tumors, bone metastasis is frequently associated with prostate cancer which is seen in about 80% of patients. During cancer treatments, some tumor cells switch to a "dormant mode" to help tumor cells avoid attack from the immune system and anti-tumor therapies. In this dormant mode, tumor cells can be resuscitated, causing cancer to reoccur. The generally accepted explanation for this phenomenon is that the tumor cells have spread to the bone marrow before treatment and are dormant in the bone marrow. However, the key mechanism for inducing and maintaining the dormancy of these prostate cancer disseminated tumor cells in the bone marrow is still unclear. Therefore, studying the dormancy mechanism of tumor cells in bone metastasis is of great significance for the treatment and the prevention of recurrence of prostate cancer.MethodsWe obtained single-cell RNA-seq data of tumors from mouse models of prostate cancer bone metastasis mouse model numbered (GSE147150) from the GEO database, and obtained RNA-seq expression data and clinical information from The Cancer Genome Atlas Program (TCGA) of prostate cancer patients from the USCS Xena database. Screening of differential genes and annotation of GO functions were performed separately. Subsequently, the screened differential genes were compared and analyzed with 50 classic Hallmark signaling pathways, and the prognosis analysis of prostate cancer patients in TCGA data was performed to discover the key genes of the dormant mechanism of tumor cells in bone metastasis, and obtain new biomarkers that can be used to predict the prognosis of patients.ResultsA total of 378 differentially expressed genes were screened, of which 293 were significantly up-regulated and 85 were significantly down-regulated. Among them, the up-regulated genes were mainly related to the immune response, and the down-regulated genes were mainly related to the cell cycle. Through GSVA (Gene set variation analysis), it is found that there are differences in a total of 3 signal pathways: COMPLEMENT, MYC_TARGETS_V1 and MYC_TARGETS_V2. By comparing and analyzing the significantly down-regulated genes in dormant tumor cells with MYC_TARGETS_V1, MYC_TARGETS_V2, three significantly down-regulated genes were obtained: Ccna2, Mad2L1 and Plk1.ConclusionIn summary, our findings indicate that the MYC targeting gene Mad2L1 is potentially related to the dormancy mechanism of prostate cancer. At the same time, Mad2L1, a gene associated with dormant prostate cancer cells, may be used as a biomarker for prognostic survival.

Development of New Mouse Breast Cancer Model of Local Bone Metastasis and Verification Using Bisphosphonates.

Local tumor injection models require complicated procedures. The purpose was to establish a simple local bone metastasis model using normal mice, and to study the usefulness of the model with bisphosphonates (BP). This study used a versatile C57BL/6 mouse model and E0771 cells. Tumor cells were injected into the right femur. Mice were divided into groups depending on the concentration of cells injected and the use of BP or not. The degree of bone destruction between the different conditions was compared using micro-computed tomography (μCT). Bone destruction was confirmed in four mice in the high-concentration group at 3 weeks, and in all other mice at 4 and 6 weeks. At 6 weeks post-injection, bone destruction was significantly suppressed in the BP group (p<0.05). We created a breast cancer mouse model of local bone metastasis. Zoledronate showed the same usefulness as in previous models. It may be an effective model for evaluating treatments for bone metastasis.

Non-invasively Imageable Tibia-tumor-fragment Implantation Experimental-bone-metastasis Mouse Model of GFP-expressing Prostate Cancer.

Although the 5-year survival rate for localized prostate cancer is nearly 100%, prognosis for patients with metastases, of which the bone is the most common site, is poor. In order to evaluate efficacy of treatments against metastatic prostate cancer, experimental tibia-bone-metastasis mouse models of prostate cancer have been previously established. In the present study, we used a novel procedure for establishment of an experimental tibiabone metastasis mouse model, with human PC-3 prostate cancer expressing green fluorescent protein (GFP), that more closely matches prostate cancer growing in the bone. PC-3 human prostate cancer cells, labeled with GFP, were initially subcutaneously injected into the flank of five male nude mice to obtain tumor tissues. Once the tumor tissue grew larger than 10 mm in diameter, the tumor tissue was harvested and minced into fragments of 1 mm3 A 1-mm hole was made in the proximal left tibia of eight male nude mice, using the tip of a 5-mm blade, and a tumor fragment was implanted into the hole for an exact fit. Tumor size was measured once a week, by non-invasive imaging of GFP fluorescence. The mice were sacrificed four weeks after tumor implantation. Tumors grew in 8 out of 8 mice (100%). All tumors were non-invasively detectable with GFP fluorescence, through the skin. Increased tumor growth in the tibia was observed every week. The establishment in the tibia of the novel experimental bone-metastatic mouse model of human prostate cancer enables facile screening, in a clinically-relevant system, of improved therapeutics for this recalcitrant disease.