Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes antitumor immunity in solid cancers, but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional coactivator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend on oncogene-mediated ECM composition and remodeling.

Collagen VI deposition mediates stromal T cell trapping through inhibition of T cell motility in the prostate tumor microenvironment.

The tumor extracellular matrix (ECM) is a barrier to anti-tumor immunity in solid tumors by disrupting T cell-tumor cell interaction underlying the need for elucidating mechanisms by which specific ECM proteins impact T cell motility and activity within the desmoplastic stroma of solid tumors. Here, we show that Collagen VI (Col VI) deposition correlates with stromal T cell density in human prostate cancer specimens. Furthermore, motility of CD4+ T cells is completely ablated on purified Col VI surfaces when compared with Fibronectin and Collagen I. Importantly, T cells adhered to Col VI surfaces displayed reduced cell spreading and fibrillar actin, indicating a reduction in traction force generation accompanied by a decrease in integrin ß1 clustering. We found that CD4+ T cells largely lack expression of integrin α1 in the prostate tumor microenvironment and that blockade of α1ß1 integrin heterodimers inhibited CD8+ T cell motility on prostate fibroblast-derived matrix, while re-expression of ITGA1 improved motility. Taken together, we show that the Col VI-rich microenvironment in prostate cancer reduces the motility of CD4+ T cells lacking integrin α1, leading to their accumulation in the stroma, thus putatively inhibiting anti-tumor T cell responses.

Nanoparticle-based modulation of CD4+ T cell effector and helper functions enhances adoptive immunotherapy.

Helper (CD4+) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8+) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4+ T cells hinders consistency and scalability of CD4+ T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4+ T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4+ T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8+ T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4+ T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4+ T cell responses.

Disruption of STAT5A and NMI signaling axis leads to ISG20-driven metastatic mammary tumors.

Molecular dynamics of developmental processes are repurposed by cancer cells to support cancer initiation and progression. Disruption of the delicate balance between cellular differentiation and plasticity during mammary development leads to breast cancer initiation and metastatic progression. STAT5A is essential for differentiation of secretory mammary alveolar epithelium. Active STAT5A characterizes breast cancer patients for favorable prognosis. N-Myc and STAT Interactor protein (NMI) was initially discovered as a protein that interacts with various STATs; however, the relevance of these interactions to normal mammary development and cancer was not known. We observe that NMI protein is expressed in the mammary ductal epithelium at the onset of puberty and is induced in pregnancy. NMI protein is decreased in 70% of patient specimens with metastatic breast cancer compared to primary tumors. Here we present our finding that NMI and STAT5A cooperatively mediate normal mammary development. Loss of NMI in vivo caused a decrease in STAT5A activity in normal mammary epithelial as well as breast cancer cells. Analysis of STAT5A mammary specific controlled genetic program in the context of NMI knockout revealed ISG20 (interferon stimulated exonuclease gene 20, a protein involved in rRNA biogenesis) as an unfailing negatively regulated target. Role of ISG20 has never been described in metastatic process of mammary tumors. We observed that overexpression of ISG20 is increased in metastases compared to matched primary breast tumor tissues. Our observations reveal that NMI-STAT5A mediated signaling keeps a check on ISG20 expression via miR-17-92 cluster. We show that uncontrolled ISG20 expression drives tumor progression and metastasis.

Hypoxia re-programs 2'-O-Me modifications on ribosomal RNA.

Hypoxia is one of the critical stressors encountered by various cells of the human body under diverse pathophysiologic conditions including cancer and has profound impacts on several metabolic and physiologic processes. Hypoxia prompts internal ribosome entry site (IRES)-mediated translation of key genes, such as VEGF, that are vital for tumor progression. Here, we describe that hypoxia remarkably upregulates RNA Polymerase I activity. We discovered that in hypoxia, rRNA shows a different methylation pattern compared to normoxia. Heterogeneity in ribosomes due to the diversity of ribosomal RNA and protein composition has been postulated to generate "specialized ribosomes" that differentially regulate translation. We find that in hypoxia, a sub-set of differentially methylated ribosomes recognizes the VEGF-C IRES, suggesting that ribosomal heterogeneity allows for altered ribosomal functions in hypoxia.

Hydrogel Network Dynamics Regulate Vascular Morphogenesis.

Matrix dynamics influence how individual cells develop into complex multicellular tissues. Here, we develop hydrogels with identical polymer components but different crosslinking capacities to enable the investigation of mechanisms underlying vascular morphogenesis. We show that dynamic (D) hydrogels increase the contractility of human endothelial colony-forming cells (hECFCs), promote the clustering of integrin ß1, and promote the recruitment of vinculin, leading to the activation of focal adhesion kinase (FAK) and metalloproteinase expression. This leads to the robust assembly of vasculature and the deposition of new basement membrane. We also show that non-dynamic (N) hydrogels do not promote FAK signaling and that stiff D- and N-hydrogels are constrained for vascular morphogenesis. Furthermore, D-hydrogels promote hECFC microvessel formation and angiogenesis in vivo. Our results indicate that cell contractility mediates integrin signaling via inside-out signaling and emphasizes the importance of matrix dynamics in vascular tissue formation, thus informing future studies of vascularization and tissue engineering applications.

Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

Lymphocyte motility is governed by a complex array of mechanisms, and highly dependent on external microenvironmental cues. Tertiary lymphoid sites in particular have unique physical structure such as collagen fiber alignment, due to matrix deposition and remodeling. Three dimensional studies of human lymphocytes in such environments are lacking. We hypothesized that aligned collagenous environment modulates CD8+ T cells motility. We encapsulated activated CD8+ T cells in collagen hydrogels of distinct fiber alignment, a characteristic of tumor microenvironments. We found that human CD8+ T cells move faster and more persistently in aligned collagen fibers compared with nonaligned collagen fibers. Moreover, CD8+ T cells move along the axis of collagen alignment. We showed that myosin light chain kinase (MLCK) inhibition could nullify the effect of aligned collagen on CD8+ T cell motility patterns by decreasing T cell turning in unaligned collagen fiber gels. Finally, as an example of a tertiary lymphoid site, we found that xenograft prostate tumors exhibit highly aligned collagen fibers. We observed CD8+ T cells alongside aligned collagen fibers, and found that they are mostly concentrated in the periphery of tumors. Overall, using an in vitro controlled hydrogel system, we show that collagen fiber organization modulates CD8+ T cells movement via MLCK activation thus providing basis for future studies into relevant therapeutics.

Engineering an Artificial T-Cell Stimulating Matrix for Immunotherapy.

T cell therapies require the removal and culture of T cells ex vivo to expand several thousand-fold. However, these cells often lose the phenotype and cytotoxic functionality for mediating effective therapeutic responses. The extracellular matrix (ECM) has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies. Here, a hyaluronic acid (HA)-based hydrogel is engineered to present the two stimulatory signals required for T-cell activation-termed an artificial T-cell stimulating matrix (aTM). It is found that biophysical properties of the aTM-stimulatory ligand density, stiffness, and ECM proteins-potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM results in a rapid and robust expansion of rare, antigen-specific CD8+ T cells. Adoptive transfer of these tumor-specific cells significantly suppresses tumor growth and improves animal survival compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM-mimetic materials for therapeutic immune stimulation in the future.

Hypoxia and matrix viscoelasticity sequentially regulate endothelial progenitor cluster-based vasculogenesis.

Vascular morphogenesis is the formation of endothelial lumenized networks. Cluster-based vasculogenesis of endothelial progenitor cells (EPCs) has been observed in animal models, but the underlying mechanism is unknown. Here, using O2-controllabe hydrogels, we unveil the mechanism by which hypoxia, co-jointly with matrix viscoelasticity, induces EPC vasculogenesis. When EPCs are subjected to a 3D hypoxic gradient ranging from <2 to 5%, they rapidly produce reactive oxygen species that up-regulate proteases, most notably MMP-1, which degrade the surrounding extracellular matrix. EPC clusters form and expand as the matrix degrades. Cell-cell interactions, including those mediated by VE-cadherin, integrin-ß2, and ICAM-1, stabilize the clusters. Subsequently, EPC sprouting into the stiffer, intact matrix leads to vascular network formation. In vivo examination further corroborated hypoxia-driven clustering of EPCs. Overall, this is the first description of how hypoxia mediates cluster-based vasculogenesis, advancing our understanding toward regulating vascular development as well as postnatal vasculogenesis in regeneration and tumorigenesis.

A Feedback Loop between Hypoxia and Matrix Stress Relaxation Increases Oxygen-Axis Migration and Metastasis in Sarcoma.

Upregulation of collagen matrix crosslinking directly increases its ability to relieve stress under the constant strain imposed by solid tumor, a matrix property termed stress relaxation. However, it is unknown how rapid stress relaxation in response to increased strain impacts disease progression in a hypoxic environment. Previously, it has been demonstrated that hypoxia-induced expression of the crosslinker procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), in sarcomas has resulted in increased lung metastasis. Here, we show that short stress relaxation times led to increased cell migration along a hypoxic gradient in 3D collagen matrices, and rapid stress relaxation upregulated PLOD2 expression via TGFß-SMAD2 signaling, forming a feedback loop between hypoxia and the matrix. Inhibition of this pathway led to a decrease in migration along the hypoxic gradients. In vivo, sarcoma primed in a hypoxic matrix with short stress relaxation time enhanced collagen fiber size and tumor density and increased lung metastasis. High expression of PLOD2 correlated with decreased overall survival in patients with sarcoma. Using a patient-derived sarcoma cell line, we developed a predictive platform for future personalized studies and therapeutics. Overall, these data show that the interplay between hypoxia and matrix stress relaxation amplifies PLOD2, which in turn accelerates sarcoma cell motility and metastasis. SIGNIFICANCE: These findings demonstrate that mechanical (stress relaxation) and chemical (hypoxia) properties of the tumor microenvironment jointly accelerate sarcoma motility and metastasis via increased expression of collagen matrix crosslinker PLOD2.

Conditional knockout of N-Myc and STAT interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors.

The process of organ development requires a delicate balance between cellular plasticity and differentiation. This balance is disrupted in cancer initiation and progression. N-Myc and STAT interactor (NMI: human or Nmi: murine) has emerged as a relevant player in the etiology of breast cancer. However, a fundamental understanding of its relevance to normal mammary biology is lacking. To gain insight into its normal function in mammary gland, we generated a mammary-specific Nmi knockout mouse model. We observed that Nmi protein expression is induced in mammary epithelium at the onset of pregnancy, in luminal cells and persists throughout lactation. Nmi knockout results in a precocious alveolar phenotype. These alveoli exhibit an extensive presence of nuclear ß-catenin and enhanced Wnt/ß-catenin signaling. The Nmi knockout pubertal ductal tree shows enhanced invasion of the mammary fatpad and increased terminal end bud numbers. Tumors from Nmi null mammary epithelium show a significant enrichment of poorly differentiated cells with elevated stem/progenitor markers, active Wnt/ß-catenin signaling, highly invasive morphology as well as, increased number of distant metastases. Our study demonstrates that Nmi has a distinct role in the differentiation process of mammary luminal epithelial cell compartment and developmental aberrations resulting from Nmi absence contribute to metastasis and demonstrates that aberration in normal developmental program can lead to metastatic disease, highlighting the contribution and importance of luminal progenitor cells in driving metastatic disease.

Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model.

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

Developing Cures: Targeting Ontogenesis in Cancer.

Cancer has long been known to histologically resemble developing embryonic tissue. Since this early observation, a mounting body of evidence suggests that cancer mimics or co-opts developmental processes to facilitate tumor initiation and progression. Programs important in both normal ontogenesis and cancer progression broadly fall into three domains: the lineage commitment of pluripotent stem cells, the appropriation of primordial mechanisms of cell motility and invasion, and the influence of multiple aspects of the microenvironment on the parenchyma. In this review we discuss how derangements in these developmental pathways drive cancer progression with a particular focus on how they have emerged as targets of novel treatment strategies.

BH3 mimetics suppress CXCL12 expression in human malignant peripheral nerve sheath tumor cells.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Schwann cell-derived neoplasms of the peripheral nervous system that have recently been shown to possess an autocrine CXCL12/CXCR4 signaling loop that promotes tumor cell proliferation and survival. Importantly, the CXCL12/CXCR4 signaling axis is driven by availability of the CXCL12 ligand rather than CXCR4 receptor levels alone. Therefore, pharmacological reduction of CXCL12 expression could be a potential chemotherapeutic target for patients with MPNSTs or other pathologies wherein the CXCL12/CXCR4 signaling axis is active. AT101 is a well-established BCL-2 homology domain 3 (BH3) mimetic that we recently demonstrated functions as an iron chelator and thus acts as a hypoxia mimetic. In this study, we found that AT101 significantly reduces CXCL12 mRNA and secreted protein in established human MPNST cell lines in vitro. This effect was recapitulated by other BH3 mimetics [ABT-737 (ABT), obatoclax (OBX) and sabutoclax (SBX)] but not by desferrioxamine (DFO), an iron chelator and known hypoxia mimetic. These data suggest that CXCL12 reduction is a function of AT101's BH3 mimetic property rather than its iron chelation ability. Additionally, this study investigates a potential mechanism of BH3 mimetic-mediated CXCL12 suppression: liberation of a negative CXCL12 transcriptional regulator, poly (ADP-Ribose) polymerase I (PARP1) from its physical interaction with BCL-2. These data suggest that clinically available BH3 mimetics might prove therapeutically useful at least in part by virtue of their ability to suppress CXCL12 expression.

Roles of N-Myc and STAT interactor in cancer: From initiation to dissemination.

N-myc & STAT Interactor, NMI, is a protein that has mostly been studied for its physical interactions with transcription factors that play critical roles in tumor growth, progression and metastasis. NMI is an inducible protein, thus its intracellular levels and location can vary dramatically, influencing a diverse array of cellular functions in a context-dependent manner. The physical interactions of NMI with its binding partners have been linked to many aspects of tumor biology including DNA damage response, cell death, epithelial-to-mesenchymal transition and stemness. Thus, discovering more details about the function(s) of NMI could reveal key insights into how transcription factors like c-Myc, STATs and BRCA1 are contextually regulated. Although a normal, physiological function of NMI has not yet been discovered, it has potential roles in pathologies ranging from viral infection to cancer. This review provides a timely perspective of the unfolding roles of NMI with specific focus on cancer progression and metastasis.

microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer.

BACKGROUND: N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis. METHOD: Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3ß phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses. RESULTS: Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/ß-catenin signaling resulting from inactivation of GSK3ß. CONCLUSION: Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells.

BACKGROUND: Interleukin (IL)-11, a cytokine produced by breast cancer, has been implicated in breast cancer-induced osteolysis (bone destruction) but the mechanism(s) of action remain controversial. Some studies show that IL-11 is able to promote osteoclast formation independent of the receptor activator of NF-κB ligand (RANKL), while others demonstrate IL-11 can induce osteoclast formation by inducing osteoblasts to secrete RANKL. This work aims to further investigate the role of IL-11 in metastasis-induced osteolysis by addressing a new hypothesis that IL-11 exerts effects on osteoclast progenitor cells. METHODS: To address the precise role of breast cancer-derived IL-11 in osteoclastogenesis, we determined the effect of breast cancer conditioned media on osteoclast progenitor cells with or without an IL-11 neutralizing antibody. We next investigated whether recombinant IL-11 exerts effects on osteoclast progenitor cells and survival of mature osteoclasts. Finally, we examined the ability of IL-11 to mediate osteoclast formation in tissue culture dishes and on bone slices in the absence of RANKL, with suboptimal levels of RANKL, or from RANKL-pretreated murine bone marrow macrophages (BMMs). RESULTS: We found that freshly isolated murine bone marrow cells cultured in the presence of breast cancer conditioned media for 6 days gave rise to a population of cells which were able to form osteoclasts upon treatment with RANKL and M-CSF. Moreover, a neutralizing anti-IL-11 antibody significantly inhibited the ability of breast cancer conditioned media to promote the development and/or survival of osteoclast progenitor cells. Similarly, recombinant IL-11 was able to sustain a population of osteoclast progenitor cells. However, IL-11 was unable to exert any effect on osteoclast survival, induce osteoclastogenesis independent of RANKL, or promote osteoclastogenesis in suboptimal RANKL conditions. CONCLUSIONS: Our data indicate that a) IL-11 plays an important role in osteoclastogenesis by stimulating the development and/or survival of osteoclast progenitor cells and b) breast cancer may promote osteolysis in part by increasing the pool of osteoclast progenitor cells via tumor cell-derived IL-11. However, given the heterogeneous nature of the bone marrow cells, the precise mechanism by which IL-11 treatment gives rise to a population of osteoclast progenitor cells warrants further investigation.