Developing a definition of immune exclusion in cancer: results of a modified Delphi workshop.

Checkpoint inhibitors represent an effective treatment approach for a variety of cancers through their inhibition of immune regulatory pathways within the tumor microenvironment (TME). Unfortunately only a minority of patients with cancer achieve clinical benefit from immunotherapy, with the TME emerging as an important predictor of outcomes and sensitivity to therapy. The extent and pattern of T-cell infiltration can vary prominently within/across tumors and represents a biological continuum. Three immune profiles have been identified along this continuum: 'immune-desert' or 'T-cell cold' phenotype, 'immune-active', 'inflamed', or 'T-cell hot' phenotype, and 'immune excluded' phenotype. Of the three profiles, immune excluded remains the most ill-defined with no clear, universally accepted definition even though it is commonly associated with lack of response to immune checkpoint inhibitors and poor clinical outcomes. To address this, 16 multidisciplinary cancer experts from around the world were invited to participate in a symposium using a three-round modified Delphi approach. The first round was an open-ended questionnaire distributed via email and the second was an in-person discussion of the first round results that allowed for statements to be revised as necessary to achieve a maximum consensus (75% agreement) among the rating committee (RC). The final round questionnaire was distributed to the RC via email and had a 100% completion rate. The Delphi process resulted in moving us closer to a consensus definition for immune exclusion that is practical, clinically pertinent, and applicable across a wide range of cancer histologies. A general consensus of the role of immune exclusion in resistance to checkpoint therapy and five research priorities emerged from this process. Together, these tools could help efforts designed to address the underlying mechanisms of immune exclusion that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms to improve patient outcomes.

Unusual Suspects: Bone and Cartilage ECM Proteins as Carcinoma Facilitators.

The extracellular matrix (ECM) is the complex three-dimensional network of fibrous proteins and proteoglycans that constitutes an essential part of every tissue to provide support for normal tissue homeostasis. Tissue specificity of the ECM in its topology and structure supports unique biochemical and mechanical properties of each organ. Cancers, like normal tissues, require the ECM to maintain multiple processes governing tumor development, progression and spread. A large body of experimental and clinical evidence has now accumulated to demonstrate essential roles of numerous ECM components in all cancer types. Latest findings also suggest that multiple tumor types express, and use to their advantage, atypical ECM components that are not found in the cancer tissue of origin. However, the understanding of cancer-specific expression patterns of these ECM proteins and their exact roles in selected tumor types is still sketchy. In this review, we summarize the latest data on the aberrant expression of bone and cartilage ECM proteins in epithelial cancers and their specific functions in the pathogenesis of carcinomas and discuss future directions in exploring the utility of this selective group of ECM components as future drug targets.

Differentiation of the tumor microenvironment: are CAFs the Organizer?

Cancers contain a suite of genetically stable cells within an extracellular matrix, collectively termed the tumor microenvironment (TME). The TME strongly influences disease outcome for patients. Gleaning clues from the literature, we propose that the TME should be viewed not as disparate populations of cells constituting a pathological lesion, but as a cohesive tissue constituting a novel pathological organ, arising from the coordinated differentiation of its constituent cell types - a process we have termed tumor-associated neodifferentiation (TAND). We also discuss why cancer-associated fibroblasts (CAFs) may assume the role of Organizer of this organ, directing the recruitment and differentiation of cells within the TME. Viewing the microenvironment in this way will reveal new cancer vulnerabilities that may be exploited for therapy.

CXCR4-CCR7 Heterodimerization Is a Driver of Breast Cancer Progression.

Metastatic breast cancer has one of the highest mortality rates among women in western society. Chemokine receptors CXCR4 and CCR7 have been shown to be linked to the metastatic spread of breast cancer, however, their precise function and underlying molecular pathways leading to the acquisition of the pro-metastatic properties remain poorly understood. We demonstrate here that the CXCR4 and CCR7 receptor ligands, CXCL12 and CCL19, cooperatively bind and selectively elicit synergistic signalling responses in invasive breast cancer cell lines as well as primary mammary human tumour cells. Furthermore, for the first time, we have documented the presence of CXCR4-CCR7 heterodimers in advanced primary mammary mouse and human tumours where number of CXCR4-CCR7 complexes directly correlate with the severity of the disease. The functional significance of the CXCR4-CCR7 association was also demonstrated when their forced heterodimerization led to the acquisition of invasive phenotype in non-metastatic breast cancer cells. Taken together, our data establish the CXCR4-CCR7 receptor complex as a new functional unit, which is responsible for the acquisition of breast cancer cell metastatic phenotype and which may serve as a novel biomarker for invasive mammary tumours.

Publisher Correction: ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism.

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

ACKR4 restrains antitumor immunity by regulating CCL21.

Current immunotherapies involving CD8+ T cell responses show remarkable promise, but their efficacy in many solid tumors is limited, in part due to the low frequency of tumor-specific T cells in the tumor microenvironment (TME). Here, we identified a role for host atypical chemokine receptor 4 (ACKR4) in controlling intratumor T cell accumulation and activation. In the absence of ACKR4, an increase in intratumor CD8+ T cells inhibited tumor growth, and nonhematopoietic ACKR4 expression was critical. We show that ACKR4 inhibited CD103+ dendritic cell retention in tumors through regulation of the intratumor abundance of CCL21. In addition, preclinical studies indicate that ACKR4 and CCL21 are potential therapeutic targets to enhance responsiveness to immune checkpoint blockade or T cell costimulation.

The Role of the Extracellular Matrix and Its Molecular and Cellular Regulators in Cancer Cell Plasticity.

The microenvironment encompasses all components of a tumor other than the cancer cells themselves. It is highly heterogenous, comprising a cellular component that includes immune cells, fibroblasts, adipocytes, and endothelial cells, and a non-cellular component, which is a meshwork of polymeric proteins and accessory molecules, termed the extracellular matrix (ECM). The ECM provides both a biochemical and biomechanical context within which cancer cells exist. Cancer progression is dependent on the ability of cancer cells to traverse the ECM barrier, access the circulation and establish distal metastases. Communication between cancer cells and the microenvironment is therefore an important aspect of tumor progression. Significant progress has been made in identifying the molecular mechanisms that enable cancer cells to subvert the immune component of the microenvironment to facilitate tumor growth and spread. While much less is known about how the tumor cells adapt to changes in the ECM nor indeed how they influence ECM structure and composition, the importance of the ECM to cancer progression is now well established. Plasticity refers to the ability of cancer cells to modify their physiological characteristics, permitting them to survive hostile microenvironments and resist therapy. Examples include the acquisition of stemness characteristics and the epithelial-mesenchymal and mesenchymal-epithelial transitions. There is emerging evidence that the biochemical and biomechanical properties of the ECM influence cancer cell plasticity and vice versa. Outstanding challenges for the field remain the identification of the cellular mechanisms by which cancer cells establish tumor-promoting ECM characteristics and delineating the key molecular mechanisms underlying ECM-induced cancer cell plasticity. Here we summarize the current state of understanding about the relationships between cancer cells and the main stromal cell types of the microenvironment that determine ECM characteristics, and the key molecular pathways that govern this three-way interaction to regulate cancer cell plasticity. We postulate that a comprehensive understanding of this dynamic system will be required to fully exploit opportunities for targeting the ECM regulators of cancer cell plasticity.

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis.

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation phase of mRNA translation and hence protein synthesis. Increasing evidence indicates that eEF2K plays an important role in the survival and migration of cancer cells and in tumor progression. As demonstrated by two-dimensional wound-healing and three-dimensional transwell invasion assays, knocking down or inhibiting eEF2K in cancer cells impairs migration and invasion of cancer cells. Conversely, exogenous expression of eEF2K or knocking down eEF2 (the substrate of eEF2K) accelerates wound healing and invasion. Importantly, using LC-HDMSE analysis, we identify 150 proteins whose expression is decreased and 73 proteins which are increased upon knocking down eEF2K in human lung carcinoma cells. Of interest, 34 downregulated proteins are integrins and other proteins implicated in cell migration, suggesting that inhibiting eEF2K may help prevent cancer cell mobility and metastasis. Interestingly, eEF2K promotes the association of integrin mRNAs with polysomes, providing a mechanism by which eEF2K may enhance their cellular levels. Consistent with this, genetic knock down or pharmacological inhibition of eEF2K reduces the protein expression levels of integrins. Notably, pharmacological or genetic inhibition of eEF2K almost completely blocked tumor growth and effectively prevented the spread of tumor cells in vivo. High levels of eEF2K expression were associated with invasive carcinoma and metastatic tumors. These data provide the evidence that eEF2K is a new potential therapeutic target for preventing tumor metastasis.

Interplay between CCR7 and Notch1 axes promotes stemness in MMTV-PyMT mammary cancer cells.

BACKGROUND: Breast cancer is the major cause of cancer-related mortality in women. It is thought that quiescent stem-like cells within solid tumors are responsible for cancer maintenance, progression and eventual metastasis. We recently reported that the chemokine receptor CCR7, a multi-functional regulator of breast cancer, maintains the stem-like cell population. METHODS: This study used a combination of molecular and cellular assays on primary mammary tumor cells from the MMTV-PyMT transgenic mouse with or without CCR7 to examine the signaling crosstalk between CCR7 and Notch pathways. RESULTS: We show for the first time that CCR7 functionally intersects with the Notch signaling pathway to regulate mammary cancer stem-like cells. In this cell subpopulation, CCR7 stimulation activated the Notch signaling pathway, and deletion of CCR7 significantly reduced the levels of activated cleaved Notch1. Moreover, blocking Notch activity prevented specific ligand-induced signaling of CCR7 and augmentation of mammary cancer stem-like cell function. CONCLUSION: Crosstalk between CCR7 and Notch1 promotes stemness in mammary cancer cells and may ultimately potentiate mammary tumor progression. Therefore, dual targeting of both the CCR7 receptor and Notch1 signaling axes may be a potential therapeutic avenue to specifically inhibit the functions of breast cancer stem cells.

Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein.

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models. Birth Defects Research (Part A) 106:636-646, 2016. © 2016 Wiley Periodicals, Inc.

The chemokine receptor CCR6 facilitates the onset of mammary neoplasia in the MMTV-PyMT mouse model via recruitment of tumor-promoting macrophages.

BACKGROUND: The expression of the chemokine receptor CCR6 has been previously correlated with higher grades and stages of breast cancer and decreased relapse-free survival. Also, its cognate chemokine ligand CCL20 has been reported to induce proliferation of cultured human breast epithelial cells. METHODS: To establish if CCR6 plays a functional role in mammary tumorigenesis, a bigenic MMTV-PyMT CCR6-null mouse was generated and mammary tumor development was assessed. Levels of tumor-infiltrating immune cells within tumor-bearing mammary glands from MMTV-PyMT Ccr6 (WT) and Ccr6 (-/-) mice were also analyzed. RESULTS: Deletion of CCR6 delayed tumor onset, significantly reduced the extent of initial hyperplastic outgrowth, and decreased tumor incidence in PyMT transgenic mice. CCR6 was then shown to promote the recruitment of pro-tumorigenic macrophages to the tumor site, facilitating the onset of neoplasia. CONCLUSIONS: This study delineated for the first time a role for CCR6 in the development of breast cancer, and demonstrated a critical function for this receptor in maintaining the pro-tumorigenic cancer microenvironment.

Breast cancer stem cells and the immune system: promotion, evasion and therapy.

Cancer stem cells are believed to be a subset of heterogeneous tumour cells responsible for tumour initiation, growth, local invasion, and metastasis. In breast cancer, numerous factors have been implicated in regulation of cancer stem cells, but there is still a paucity of information regarding precise molecular and cellular mechanisms guiding their pathobiology. Components of both the adaptive and the innate immune system have been shown to play a crucial role in supporting breast cancer growth and spread, and recently some immune mediators, both molecules and cells, have been reported to influence breast cancer stem cell biology. This review summarises a small, pioneering body of evidence for the potentially important function of the "immuniche" in maintaining and supporting breast cancer stem cells.

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model.

INTRODUCTION: Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer. METHODS: Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4. RESULTS: In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. CONCLUSION: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.

Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells.

CXCR4 is a G protein-coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein alpha and beta subunits to form a heterotrimeric complex with CXCR4. Galpha and Gbeta were able to bind to CXCR4 independently in all cell lines, but the association of G protein alphabetagamma heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein-coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein-receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells.

In the multimolecular environment in tissues and organs, cross-talk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDA-MB-231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting that CXCR4 and G proteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and the G protein subunits, G(i)alpha2 and Gbeta, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of G(i)alpha2 and Gbeta from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, G(i)alpha2 and Gbeta.

Sequencing, transcript identification, and quantitative gene expression profiling in the breast cancer loss of heterozygosity region 16q24.3 reveal three potential tumor-suppressor genes.

Loss of heterozygosity (LOH) of chromosome 16q24.3 is a common genetic alteration observed in invasive ductal and lobular breast carcinomas. We constructed a physical map and generated genomic DNA sequence data spanning 2.4 Mb in this region. Detailed in silico and in vitro analyses of the genomic sequence data enabled the identification of 104 genes. It was hypothesized that tumor-suppressor genes would exhibit marked mRNA expression variability in a panel of breast cancer cell lines as a result of downregulation due to mutation or hypermethylation. We examined the mRNA expression profiles of the genes identified at 16q24.3 in normal breast, a normal breast epithelial cell line, and several breast cancer cell lines exhibiting 16q24.3 LOH. Three of the genes, CYBA, Hs.7970, and CBFA2T3, exhibited variability ten times higher than the baseline. The possible role of these genes as tumor suppressors is discussed.

CBFA2T3 (MTG16) is a putative breast tumor suppressor gene from the breast cancer loss of heterozygosity region at 16q24.3.

Numerous cytogenetic and molecular studies of breast cancer have identified frequent loss of heterozygosity (LOH) of the long arm of human chromosome 16. On the basis of these data, the likely locations of breast cancer tumor suppressor genes are bands 16q22.1 and 16q24.3. We have mapped the CBFA2T3 (MTG16) gene, previously cloned as a fusion partner of the AML1 protein from a rare (16;21) leukemia translocation, to the 16q24.3 breast cancer LOH region. The expression of CBFA2T3 was significantly reduced in a number of breast cancer cell lines and in primary breast tumors, including early ductal carcinomas in situ, when compared with nontransformed breast epithelial cell lines and normal breast tissue. Reintroduction of CBFA2T3 into different breast tumor derived cell lines with decreased expression of this gene reduced colony growth on plastic and in soft agar. CBFA2T3 was shown to function as a transcriptional repressor when tethered to the GAL4 DNA-binding domain in a reporter gene assay and, therefore, has the potential to be a transcriptional repressor in normal breast epithelial cells. Taken together, these findings suggest that CBFA2T3 is a likely candidate for the breast cancer tumor suppressor gene that is the target for the frequent 16q24 LOH in breast neoplasms.