Arginase inhibition suppresses lung metastasis in the 4T1 breast cancer model independently of the immunomodulatory and anti-metastatic effects of VEGFR-2 blockade.

Tumor angiogenesis promotes tumor growth and metastasis. Anti-angiogenic therapy in combination with chemotherapy is used for the treatment of metastatic cancers, including breast cancer but therapeutic benefits are limited. Mobilization and accumulation of myeloid-derived suppressor cells (MDSC) during tumor progression and therapy have been implicated in metastasis formation and resistance to anti-angiogenic treatments. Here, we used the 4T1 orthotopic syngenic mouse model of mammary adenocarcinoma to investigate the effect of VEGF/VEGFR-2 axis inhibition on lung metastasis, MDSC and regulatory T cells (Tregs). We show that treatment with the anti-VEGFR-2 blocking antibody DC101 inhibits primary tumor growth, angiogenesis and lung metastasis. DC101 treatment had no effect on MDSC mobilization, but partially attenuated the inhibitory effect of mMDSC on T cell proliferation and decreased the frequency of Tregs in primary tumors and lung metastases. Strikingly, DC101 treatment induced the expression of the immune-suppressive molecule arginase I in mMDSC. Treatment with the arginase inhibitor Nω-hydroxy-nor-Arginine (Nor-NOHA) reduced the inhibitory effect of MDSC on T cell proliferation and inhibited number and size of lung metastasis but had little or no additional effects in combination with DC101. In conclusion, DC101 treatment suppresses 4T1 tumor growth and metastasis, partially reverses the inhibitory effect of mMDSC on T cell proliferation, decreases Tregs in tumors and increases arginase I expression in mMDSC. Arginase inhibition suppresses lung metastasis independently of DC101 effects. These observations contribute to the further characterization of the immunomodulatory effect of anti-VEGF/VEGFR2 therapy and provide a rationale to pursue arginase inhibition as potential anti-metastatic therapy.

TLR7-based cancer immunotherapy decreases intratumoral myeloid-derived suppressor cells and blocks their immunosuppressive function.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with the capacity to inhibit immunological responses. During cancer progression, MDSC are recruited to the tumor sites and secondary lymphoid organs, leading to the suppression of the antitumor function of NK and T cells. Here, we show that the TLR7/8 agonist resiquimod (R848) has a direct effect on MDSC populations in tumor-bearing mice. Systemic application of R848 led to a rapid reduction in both intratumoral and circulating MDSC. The subpopulation of monocytic MDSC (m-MDSC) was the most affected by R848 treatment with an up to 5-fold decrease in the tumor. We found that TLR7 stimulation in tumor-bearing mice led to a maturation and differentiation of MDSC with upregulation of the surface molecules CD11c, F4/80, MHC-I, and MHC-II. MDSC treated with R848 lost their immunosuppressive function and acquired instead an antigen-presenting phenotype with the capability to induce specific T-cell proliferation. Importantly, we found that MDSC co-injected s.c. with CT26 tumor cells lost their ability to support tumor growth after pretreatment with R848. Our results demonstrate that treatment of tumor-bearing mice with a TLR7/8 agonist acts directly on MDSC to induce their maturation and leads them to acquire a non-suppressive status. Considering the obstacles posed by MDSC for cancer immunotherapy, targeting these cells by a TLR7/8 agonist may improve immune responses against cancer.

Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling.

Radiotherapy is a standard treatment after conservative breast cancer surgery. However, cancers relapsing within a previously irradiated area have an increased probability to metastasize. The mechanisms responsible for this aggressiveness remain unclear. Here, we used the clinically relevant 4T1 breast cancer model mimicking aggressive local relapse after radiotherapy to identify differences between tumors grown in untreated versus preirradiated mammary glands. Tumors grown within preirradiated beds were highly enriched in transcripts encoding collagens and other proteins building or modifying the extracellular matrix, such as laminin-332, tenascins, lysyl oxidases and matrix metalloproteinases. Type I collagen, known to directly contribute to tissue stiffening, and the pro-metastatic megakaryoblastic leukemia-1 (Mkl1) target gene tenascin-C were further investigated. Mammary tissue preirradiation induced Mkl1 nuclear translocation in the tumor cells in vivo, indicating activation of Mkl1 signaling. Transcript profiling of cultured 4T1 cells revealed that the majority of the Mkl1 target genes, including tenascin-C, required serum response factor (SRF) for their expression. However, application of dynamic strain or matrix stiffness to 4T1 cells converted the predominant SRF/Mkl1 action into SAP domain-dependent Mkl1 signaling independent of SRF, accompanied by a switch to SAP-dependent tumor cell migration. 4T1 tumors overexpressing intact Mkl1 became more metastatic within preirradiated beds, while tumors expressing Mkl1 lacking the SAP domain exhibited impaired growth and metastatic spread, and decreased Mkl1 target gene expression. Thus, we identified SAP-dependent Mkl1 signaling as a previously unrecognized mediator of aggressive progression of mammary tumors locally relapsing after radiotherapy, and provide a novel signaling pathway for therapeutic intervention.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Inhibition of the Kit ligand/c-Kit axis attenuates metastasis in a mouse model mimicking local breast cancer relapse after radiotherapy.

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy.

Molecular pathways: emerging pathways mediating growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment.

Radiotherapy is a well-established therapeutic modality in oncology. It provides survival benefits in several different cancer types. However, cancers relapsing after radiotherapy often develop into more aggressive conditions that are difficult to treat and are associated with poor prognosis. Cumulative experimental evidence indicates that the irradiated tumor bed contributes to such aggressive behavior. The involved mechanisms have for long remained elusive. Recent progress in the field revealed previously unrecognized cellular and molecular events promoting growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment. Cellular mechanisms include inhibition of sprouting angiogenesis, formation of hypoxia, activation and differentiation of stromal cells, and recruitment of bone marrow-derived cells with vasculogenic and prometastatic activities. Identified pathways include TGF-ß/ALK5, CXCL12/CXCR4, KITL/KIT, and CYR61/αVß5 integrin. The availability of pharmacologic inhibitors impinging on these pathways opens novel opportunities for translational and clinical studies. These experimental results and ongoing work highlight the importance of the irradiated microenvironment in modulating the tumor response to radiotherapy and open new opportunities for the development of novel therapeutic strategies for patients with cancer who relapse after radiotherapy. Here, we review and discuss recent advances in the field and their translational and therapeutic implications to human cancer treatment.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells.

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

The role of the BMP signaling antagonist noggin in the development of prostate cancer osteolytic bone metastasis.

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

Eight new species of the genus Micromonospora, Micromonospora citrea sp. nov., Micromonospora echinaurantiaca sp. nov., Micromonospora echinofusca sp. nov. Micromonospora fulviviridis sp. nov., Micromonospora inyonensis sp. nov., Micromonospora peucetia sp. nov., Micromonospora sagamiensis sp. nov., and Micromonospora viridifaciens sp. nov.

A previous phylogenetic study on type strains of the genus Micromonospora and Micromonospora species bearing non-validly published names has pointed towards the species status of several of latter strains. Subsequent studies on morphological, cultural, chemotaxonomic, metabolic, and genomic properties, and on whole cell mass spectrometric analyses by matrix adsorbed laser desorption/ionization time-of-flight (MALDI-TOF) confirmed the species status, leading to the proposal of eight new Micromonospora species: Micromonospora citrea sp. nov., type strain DSM 43903T, Micromonospora echinaurantiaca sp. nov., type strain DSM 43904T, Micromonospora echinofusca sp. nov., type strain DSM 43913T, Micromonospora fulviviridis sp. nov., type strain DSM 43906T, Micromonospora inyonensis sp. nov., type strain DSM 46123T, Micromonospora peucetia sp. nov., type strain DSM 43363T, Micromonospora sagamiensis sp. nov., type strain DSM 43912T and Micromonospora viridifaciens sp. nov., type strain DSM 43909T.