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Incidence of spontaneous micrometastases

Supplementary Figure S1: Spontaneous brain macrometastases analyzed by MRI or histology

Supplementary Figure S4: Detection and molecular quantification of CTCs

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood–brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359–71. ©2016 AACR.

Supplementary Figure S6: No macrometastases were detected in mice analyzed for blood vessel permeability

Supplementary Figure S5: Melanoma-derived transcripts can be found in CSF isolated from intracardiac injected mice

Supplementary Figure S7: Brain tropic melanoma cells (BT-RMS) have a more aggressive phenotype

Supplementary Figure S3: FACS analysis enables the detection and quantification of brain micrometastases

Supplementary Methods

Incidence of spontaneous brain macrometastases

Supplementary Figure S2: Molecular analysis of mCherry enables detection of micrometastases

Supplementary figure legends

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell–secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell–secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs. Cancer Res; 75(6); 963–73. ©2015 AACR.

Figure S3: Knockdown of OPN does not affect tumor cell growth in vitro.

Table 2: Summary of proteomics analysis: highest intensity extracellular proteins in tumor cell CM.

Supplementary Table 1: Conditioned medium proteome. Intensity values (Log2 values) are indicated for each proteins with and without label-free quantification (LFQ) normalization. PEP- posterior error probability. 'Extracellular' column indicates the proteins annotated as such according Gene Ontology Cellular Compartment (GOCC).

Figure S2: CD44 expression is up regulated in CAFs and in activated NMFs.

Figure S1: OPN does not affect NMFs proliferation.

Chemical element mapping is an imaging tool that provides essential information about composite materials, and it is crucial for a broad range of fields ranging from fundamental science to numerous applications. Methods that exploit x-ray fluorescence are very advantageous and are widely used, but require focusing of the input beam and raster scanning of the sample. Thus, the methods are slow and exhibit limited resolution due to focusing challenges. Here, we demonstrate an x-ray fluorescence method based on computational ghost imaging that overcomes those limitations since it does not require focusing and show that when it is combined with compressed sensing the total measurement time can be significantly reduced. Our method opens the possibility to significantly enhance the resolution of chemical element maps and to extend the applicability of x-ray fluorescence inspection to new fields where the measurement time is a critical parameter.

The outgrowth of primary melanoma, the deadliest skin cancer, and generation of metastasis is supported by the tumor microenvironment (TME) which includes non-cancerous cells. Since the TME plays an important role in melanoma pathogenesis, its targeting is a promising therapeutic approach. Thus, it is important to identify proteins in the melanoma TME that may serve as therapeutic targets. Here we show that the nicotinamide adenine dinucleotide glycohydrolase CD38 is a suitable target for this purpose. Loss of CD38 in the TME as well as inhibition of its enzymatic activity restrained outgrowth of primary melanoma generated by two transplantable models of melanoma, B16F10 and Ret-mCherry-sorted (RMS) melanoma cells. Pathological analysis indicated that loss of CD38 increased cell death and reduced the amount of cancer-associated fibroblasts (CAFs) and blood vessels. Importantly, in addition to inhibiting outgrowth of primary melanoma tumors, loss of CD38 also inhibited spontaneous occurrence of RMS pulmonary and brain metastasis. The underlying mechanism may involve, at least in the brain, inhibition of metastasis expansion, since loss of CD38 inhibited the outgrowth of B16F10 and RMS brain tumors that were generated by direct intracranial implantation. Collectively, our results suggest that targeting CD38 in the melanoma TME provides a new therapeutic approach for melanoma treatment.

// Eva Rietkotter 1 , Annalen Bleckmann 1 , Michaela Bayerlova 2 , Kerstin Menck 1 , Han-Ning Chuang 1 , Britta Wenske 1 , Hila Schwartz 3 , Neta Erez 3 , Claudia Binder 1 , Uwe-Karsten Hanisch 4 , Tobias Pukrop 1, 5 1 Department of Hematology and Medical Oncology, University Medical Center, 37075 Gottingen, Germany 2 Department of Medical Statistics, University Medical Center, 37075 Gottingen, Germany 3 Department of Pathology, Sackler School of Medicine, 69978 Tel Aviv University, Israel 4 Institute of Neuropathology, University Medical Center, 37075 Gottingen, Germany 5 Department of Hematology and Medical Oncology, University Clinic Regensburg, 93053 Regensburg, Germany Correspondence to: Tobias Pukrop, e-mail: tobias.pukrop@ukr.de Keywords: anti-CSF-1, colonization, monocyte-derived cells, microglia, metastasis Received: January 5, 2015 Accepted: April 29, 2015 Published: May 12, 2015 ABSTRACT The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown. Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression. Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood–brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359–71. ©2016 AACR.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell–secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell–secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs. Cancer Res; 75(6); 963–73. ©2015 AACR.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

Melanoma frequently metastasizes to the brain, resulting in dismal survival. A major obstacle for characterizing mechanisms of brain metastasis is the lack of tractable pre-clinical models that mimic the multi-stage process of brain metastasis. We established a novel model of spontaneous melanoma brain metastasis in immunocompetent mice. Aggressive local tumors are formed following orthotopic injection, and 3-4 months after surgical excision of the primary tumor, 50% of the mice develop brain macrometastases. By utilizing a unique ex-vivo modeling system, we detected brain micrometastases in 50-60% of the mice and quantified the metastatic load. Moreover, we show that the detection of melanoma transcripts in cerebrospinal fluid (CSF) is correlated with brain metastases. Interestingly, preliminary findings suggest that the melanoma-derived transcripts are found in exosomes. Astrocytes are glial cells that maintain brain homeostasis, and constitute a central part of the brain microenvironment. Reactive astrogliosis is the primary response of astrocytes to brain insult. While much data have accumulated on the contribution of astrogliosis to neurodegenerative diseases, the functional role of astrogliosis in promoting melanoma brain metastases is unknown. Utilizing our model we demonstrate early changes in the microenvironment and show that micrometastases are associated with activation of astrocytes and with hyperpermeability of the blood-brain-barrier. In-vitro, paracrine signaling by melanoma cells activated astrocytes to up-regulate a ‘wound healing program’ including Cxcl10 and Serpins. Reciprocally, astrocytes facilitated the invasion of melanoma cells and were necessary for primary outgrowth in 3D co-cultures. Finally, we show that intracranial co-injection of astrocytes with melanoma cells resulted in a 9 fold increase of tumor volume, and that the same gene-signature was up-regulated in astrocytes in-vivo. Studying spontaneous melanoma brain metastasis in a clinically relevant setting enables in-depth understating of the metastatic microenviromet which is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Citation Format: Hila Schwartz, Eran Blacher, Malak Amer, Nir Livneh, Anat Klein, Dikla Ben-Shushan, Shelly Soffer, Reuven Stein, Ronit Satchi-Fainaro, Viktor Umansky, Tobias Pukrop, Neta Erez. Melanoma brain metastasis is facilitated by instigation of reactive astrogliosis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C26.