MUSTANG: Multi-sample spatial transcriptomics data analysis with cross-sample transcriptional similarity guidance.

Spatially resolved transcriptomics has revolutionized genome-scale transcriptomic profiling by providing high-resolution characterization of transcriptional patterns. Here, we present our spatial transcriptomics analysis framework, MUSTANG (MUlti-sample Spatial Transcriptomics data ANalysis with cross-sample transcriptional similarity Guidance), which is capable of performing multi-sample spatial transcriptomics spot cellular deconvolution by allowing both cross-sample expression-based similarity information sharing as well as spatial correlation in gene expression patterns within samples. Experiments on a semi-synthetic spatial transcriptomics dataset and three real-world spatial transcriptomics datasets demonstrate the effectiveness of MUSTANG in revealing biological insights inherent in the cellular characterization of tissue samples under study.

Charting Single Cell Lineage Dynamics and Mutation Networks via Homing CRISPR.

Single cell lineage tracing, essential for unraveling cellular dynamics in disease evolution is critical for developing targeted therapies. CRISPR-Cas9, known for inducing permanent and cumulative mutations, is a cornerstone in lineage tracing. The novel homing guide RNA (hgRNA) technology enhances this by enabling dynamic retargeting and facilitating ongoing genetic modifications. Charting these mutations, especially through successive hgRNA edits, poses a significant challenge. Our solution, LINEMAP, is a computational framework designed to trace and map these mutations with precision. LINEMAP meticulously discerns mutation alleles at single-cell resolution and maps their complex interrelationships through a mutation evolution network. By utilizing a Markov Process model, we can predict mutation transition probabilities, revealing potential mutational routes and pathways. Our reconstruction algorithm, anchored in the Markov model's attributes, reconstructs cellular lineage pathways, shedding light on the cell's evolutionary journey to the minutiae of single-cell division. Our findings reveal an intricate network of mutation evolution paired with a predictive Markov model, advancing our capability to reconstruct single-cell lineage via hgRNA. This has substantial implications for advancing our understanding of biological mechanisms and propelling medical research forward.

Targeting Ribosome Biogenesis as a Novel Therapeutic Approach to Overcome EMT-related Chemoresistance in Breast Cancer.

Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer. Significance: This study uncovers the crucial involvement of ribosome biogenesis (RiBi) in the regulation of epithelial and mesenchymal state oscillations in breast cancer cells, which plays a major role in the development of chemoresistant metastasis. By proposing a novel therapeutic strategy targeting the RiBi pathway, the study offers significant potential to enhance treatment efficacy and outcomes for patients with advanced breast cancer. This approach could help overcome the limitations of current chemotherapy options and address the complex challenges posed by EMT-mediated chemoresistance.

Spatial Transcriptomics Depict Ligand-Receptor Cross-talk Heterogeneity at the Tumor-Stroma Interface in Long-Term Ovarian Cancer Survivors.

Advanced high-grade serous ovarian cancer (HGSC) is an aggressive disease that accounts for 70% of all ovarian cancer deaths. Nevertheless, 15% of patients diagnosed with advanced HGSC survive more than 10 years. The elucidation of predictive markers of these long-term survivors (LTS) could help identify therapeutic targets for the disease, and thus improve patient survival rates. To investigate the stromal heterogeneity of the tumor microenvironment (TME) in ovarian cancer, we used spatial transcriptomics to generate spatially resolved transcript profiles in treatment-naïve advanced HGSC from LTS and short-term survivors (STS) and determined the association between cancer-associated fibroblasts (CAF) heterogeneity and survival in patients with advanced HGSC. Spatial transcriptomics and single-cell RNA-sequencing data were integrated to distinguish tumor and stroma regions, and a computational method was developed to investigate spatially resolved ligand-receptor interactions between various tumor and CAF subtypes in the TME. A specific subtype of CAFs and its spatial location relative to a particular ovarian cancer cell subtype in the TME correlated with long-term survival in patients with advanced HGSC. Also, increased APOE-LRP5 cross-talk occurred at the stroma-tumor interface in tumor tissues from STS compared with LTS. These findings were validated using multiplex IHC. Overall, this spatial transcriptomics analysis revealed spatially resolved CAF-tumor cross-talk signaling networks in the ovarian TME that are associated with long-term survival of patients with HGSC. Further studies to confirm whether such cross-talk plays a role in modulating the malignant phenotype of HGSC and could serve as a predictive biomarker of patient survival are warranted. SIGNIFICANCE: Generation of spatially resolved gene expression patterns in tumors from patients with ovarian cancer surviving more than 10 years allows the identification of novel predictive biomarkers and therapeutic targets for better patient management. See related commentary by Kelliher and Lengyel, p. 1383.

Radiation-activated secretory proteins of Scgb1a1+ club cells increase the efficacy of immune checkpoint blockade in lung cancer.

Radiation therapy (RT) in combination with immune checkpoint inhibitor (ICI) represents a promising regimen for non-small cell lung cancer (NSCLC), however, the underlying mechanisms are poorly characterized. We identified a specific dose of RT that conferred tumor regression and improved survival in NSCLC models when combined with ICI. The immune-modulating functions of RT was ascribed to activated lung-resident Scgb1a1+ club cells. Importantly, mice with club cell-specific knockout of synaptosome-associated protein 23 failed to benefit from the combination treatment, indicating a pivotal role of club cell secretome. We identified 8 club cells secretory proteins, which inhibited immunosuppressive myeloid cells, reduced pro-tumor inflammation, and enhanced anti-tumor immunity. Notably, CC10, a member of club cell secretome was increased in plasma of NSCLC patients responding to the combination therapy. By revealing an immune-regulatory role of club cells, our studies have the potential to guide future clinical trials of ICI in NSCLC.

SIO: A Spatioimageomics Pipeline to Identify Prognostic Biomarkers Associated with the Ovarian Tumor Microenvironment.

Stromal and immune cells in the tumor microenvironment (TME) have been shown to directly affect high-grade serous ovarian cancer (HGSC) malignant phenotypes, however, how these cells interact to influence HGSC patients' survival remains largely unknown. To investigate the cell-cell communication in such a complex TME, we developed a SpatioImageOmics (SIO) pipeline that combines imaging mass cytometry (IMC), location-specific transcriptomics, and deep learning to identify the distribution of various stromal, tumor and immune cells as well as their spatial relationship in TME. The SIO pipeline automatically and accurately segments cells and extracts salient cellular features to identify biomarkers, and multiple nearest-neighbor interactions among tumor, immune, and stromal cells that coordinate to influence overall survival rates in HGSC patients. In addition, SIO integrates IMC data with microdissected tumor and stromal transcriptomes from the same patients to identify novel signaling networks, which would lead to the discovery of novel survival rate-modulating mechanisms in HGSC patients.

ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

Imatinib revives the therapeutic potential of metformin on ewing sarcoma by attenuating tumor hypoxic response and inhibiting convergent signaling pathways.

Ewing sarcoma (EwS) is an aggressive pediatric tumor treated with intensive cytotoxic chemotherapies. Overall survival for metastatic or relapsed disease is only 20-30%. Metformin has long been an attractive therapeutic option for EwS, but hypoxia limits its efficacy. Through a systematic integration of drug combination screening, bioinformatics analyses, functional and in vivo studies, and correlation with clinical outcome, we identified another known drug, imatinib that could augment the in vivo anti-tumor capacity of metformin by attenuating tumor hypoxic response. This drug combination regimen widely suppressed multiple dominant mechanisms in EwS genesis, growth, and metastasis, including key EWS-FLI1 downstream targets that converge into the PI3K/AKT/mTOR signaling pathway. In addition, the combination significantly enhanced inhibition on tumor cell proliferation by standard EwS chemotherapy drugs, including cyclophosphamide and ifosfamide. This suggests a potential clinical benefit of the metformin/imatinib combination by allowing the reduction in dose intensity of standard chemotherapy without compromising survival outcome and represents a potential faster track application for EwS patients.

Differential Contributions of Pre- and Post-EMT Tumor Cells in Breast Cancer Metastasis.

Metastases are responsible for the majority of breast cancer-associated deaths. The contribution of epithelial-to-mesenchymal transition (EMT) in the establishment of metastases is still controversial. To obtain in vivo evidence of EMT in metastasis, we established an EMT lineage tracing (Tri-PyMT) model, in which tumor cells undergoing EMT would irreversibly switch their fluorescent marker from RFP+ to GFP+ due to mesenchymal-specific Cre expression. Surprisingly, we found that lung metastases were predominantly derived from the epithelial compartment of breast tumors. However, concerns were raised on the fidelity and sensitivity of RFP-to-GFP switch of this model in reporting EMT of metastatic tumor cells. Here, we evaluated Tri-PyMT cells at the single-cell level using single-cell RNA-sequencing and found that the Tri-PyMT cells exhibited a spectrum of EMT phenotypes, with EMT-related genes concomitantly expressed with the activation of GFP. The fluorescent color switch in these cells precisely marked an unequivocal change in EMT status, defining the pre-EMT and post-EMT compartments within the tumor. Consistently, the pre-EMT cells played dominant roles in metastasis, while the post-EMT cells were supportive in promoting tumor invasion and angiogenesis. Importantly, the post-EMT (GFP+) cells in the Tri-PyMT model were not permanently committed to the mesenchymal phenotype; they were still capable of reverting to the epithelial phenotype and giving rise to secondary tumors, suggesting their persistent EMT plasticity. Our study addressed major concerns with the Tri-PyMT EMT lineage tracing model, which provides us with a powerful tool to investigate the dynamic EMT process in tumor biology. SIGNIFICANCE: These findings confirm the fidelity and sensitivity of the EMT lineage tracing (Tri-PyMT) model and highlight the differential contributions of pre- and post-EMT tumor cells in breast cancer metastasis.See related commentary by Bunz, p. 153.

Anticancer Immunotherapy by MFAP5 Blockade Inhibits Fibrosis and Enhances Chemosensitivity in Ovarian and Pancreatic Cancer.

PURPOSE: Recent studies demonstrate the role of the tumor microenvironment in tumor progression. However, strategies used to overcome the malignant phenotypes of cancer cells modulated by the microenvironment have not been thoroughly explored. In this study, we evaluated the therapeutic efficacy of a newly developed mAb targeting microfibril-associated protein 5 (MFAP5), which is secreted predominately by cancer-associated fibroblast (CAF), in ovarian and pancreatic cancer models.Experimental Design: MAbs were developed using human MFAP5 recombinant protein as an antigen in mice, and antibodies from hybridoma clones were evaluated for their specificity to human and murine MFAP5. An Octet RED384 system was used to determine the kinetics of binding affinity and the specificity of the antibody clones, which were followed by epitope mapping and functional characterization by in vitro assays. The therapeutic efficacy of a lead anti-MFAP5 antibody clone 130A in tumor suppression was evaluated by ovarian tumor- and pancreatic tumor-bearing mouse models. RESULTS: Three hybridoma clones, which produced antibodies with high affinity and specificity to MFAP5, were selected for functional studies. Antibody clone 130A, which recognizes a common epitope shared between human and murine MFAP5 protein, was further selected for in vivo studies. Results showed that clone 130A downregulated MFAP5-induced collagen production in CAFs, suppressed intratumoral microvessel leakiness, and enhanced paclitaxel bioavailability in both ovarian and pancreatic cancer mouse models. CONCLUSIONS: These data suggest that MFAP5 blockade using an immunologic approach inhibits fibrosis, induces tumor vessel normalization, and enhances chemosensitivity in ovarian and pancreatic cancer, and can be used as a novel therapeutic agent.

The Modulatory Role of MicroRNA-873 in the Progression of KRAS-Driven Cancers.

KRAS is one of the most frequently mutated proto-oncogenes in pancreatic ductal adenocarcinoma (PDAC) and aberrantly activated in triple-negative breast cancer (TNBC). A profound role of microRNAs (miRNAs) in the pathogenesis of human cancer is being uncovered, including in cancer therapy. Using in silico prediction algorithms, we identified miR-873 as a potential regulator of KRAS, and we investigated its role in PDAC and TNBC. We found that reduced miR-873 expression is associated with shorter patient survival in both cancers. miR-873 expression is significantly repressed in PDAC and TNBC cell lines and inversely correlated with KRAS levels. We demonstrate that miR-873 directly bound to the 3' UTR of KRAS mRNA and suppressed its expression. Notably, restoring miR-873 expression induced apoptosis; recapitulated the effects of KRAS inhibition on cell proliferation, colony formation, and invasion; and suppressed the activity of ERK and PI3K/AKT, while overexpression of KRAS rescued the effects mediated by miR-873. Moreover, in vivo delivery of miR-873 nanoparticles inhibited KRAS expression and tumor growth in PDAC and TNBC tumor models. In conclusion, we provide the first evidence that miR-873 acts as a tumor suppressor by targeting KRAS and that miR-873-based gene therapy may be a therapeutic strategy in PDAC and TNBC.

Systematic Identification of Druggable Epithelial-Stromal Crosstalk Signaling Networks in Ovarian Cancer.

BACKGROUND: Bulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma-cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer-stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer. METHODS: Transcriptome profiles from microdissected ovarian cancer-associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor-bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration-approved agent that suppresses the Smad signaling cascade, or vehicle control (9-11 mice per group). All statistical tests were two-sided. RESULTS: Activation of TGF-ß-dependent and TGF-ß-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer-bearing mice from 36 to 48 weeks (P = .04). CONCLUSIONS: Our findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer-stroma crosstalk networks, potentially leading to faster and more effective cures for cancers.

Dual Suppressive Effect of miR-34a on the FOXM1/eEF2-Kinase Axis Regulates Triple-Negative Breast Cancer Growth and Invasion.

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225-41. ©2018 AACR.

Targeting Brain-Adaptive Cancer Stem Cells Prohibits Brain Metastatic Colonization of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line-derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCß-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells.Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052-64. ©2018 AACR.

HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway.

Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.

Cancer-associated fibroblasts regulate endothelial adhesion protein LPP to promote ovarian cancer chemoresistance.

The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor-bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF-endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.

Platelets reduce anoikis and promote metastasis by activating YAP1 signaling.

Thrombocytosis is present in more than 30% of patients with solid malignancies and correlates with worsened patient survival. Tumor cell interaction with various cellular components of the tumor microenvironment including platelets is crucial for tumor growth and metastasis. Although it is known that platelets can infiltrate into tumor tissue, secrete pro-angiogenic and pro-tumorigenic factors and thereby increase tumor growth, the precise molecular interactions between platelets and metastatic cancer cells are not well understood. Here we demonstrate that platelets induce resistance to anoikis in vitro and are critical for metastasis in vivo. We further show that platelets activate RhoA-MYPT1-PP1-mediated YAP1 dephosphorylation and promote its nuclear translocation which induces a pro-survival gene expression signature and inhibits apoptosis. Reduction of YAP1 in cancer cells in vivo protects against thrombocytosis-induced increase in metastasis. Collectively, our results indicate that cancer cells depend on platelets to avoid anoikis and succeed in the metastatic process.Platelets have been associated with increased tumor growth and metastasis but the mechanistic details of this interaction are still unclear. Here the authors show that platelets improve anoikis resistance of cancer cells and increase metastasis by activating Yap through a RhoA/MYPT-PP1 pathway.

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Epithelial derived CTGF promotes breast tumor progression via inducing EMT and collagen I fibers deposition.

Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IκB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IκB signaling each prohibited the EMT and tumor progression.