Decoding breast cancer tissue-stroma interactions using species-specific sequencing.

INTRODUCTION: Decoding transcriptional effects of experimental tissue-tissue or cell-cell interactions is important; for example, to better understand tumor-stroma interactions after transplantation of human cells into mouse (xenografting). Transcriptome analysis of intermixed human and mouse cells has, however, frequently relied on the need to separate the two cell populations prior to transcriptome analysis, which introduces confounding effects on gene expression. METHODS: To circumvent this problem, we here describe a bioinformatics-based, genome-wide transcriptome analysis technique, which allows the human and mouse transcriptomes to be decoded from a mixed mouse and human cell population. The technique is based on a bioinformatic separation of the mouse and human transcriptomes from the initial mixed-species transcriptome resulting from sequencing an excised tumor/stroma specimen without prior cell sorting. RESULTS: Under stringent separation criteria, i.e., with a read misassignment frequency of 0.2 %, we show that 99 % of the genes can successfully be assigned to be of mouse or human origin, both in silico, in cultured cells and in vivo. We use a new species-specific sequencing technology-referred to as S(3) ("S-cube")-to provide new insights into the Notch downstream response following Notch ligand-stimulation and to explore transcriptional changes following transplantation of two different breast cancer cell lines (luminal MCF7 and basal-type MDA-MB-231) into mammary fat pad tissue in mice of different immunological status. We find that MCF7 and MDA-MB-231 respond differently to fat pad xenografting and the stromal response to transplantation of MCF7 and MDA-MB-231 cells was also distinct. CONCLUSIONS: In conclusion, the data show that the S(3) technology allows for faithful recording of transcriptomic changes when human and mouse cells are intermixed and that it can be applied to address a broad spectrum of research questions.

Microenvironmental control of breast cancer subtype elicited through paracrine platelet-derived growth factor-CC signaling.

Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.

Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination.

Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-ß in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.

Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.

Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

Paracrine signaling by platelet-derived growth factor-CC promotes tumor growth by recruitment of cancer-associated fibroblasts.

Cancer results from the concerted performance of malignant cells and stromal cells. Cell types populating the microenvironment are enlisted by the tumor to secrete a host of growth-promoting cues, thus upholding tumor initiation and progression. Platelet-derived growth factors (PDGF) support the formation of a prominent tumor stromal compartment by as of yet unidentified molecular effectors. Whereas PDGF-CC induces fibroblast reactivity and fibrosis in a range of tissues, little is known about the function of PDGF-CC in shaping the tumor-stroma interplay. Herein, we present evidence for a paracrine signaling network involving PDGF-CC and PDGF receptor-alpha in malignant melanoma. Expression of PDGFC in a mouse model accelerated tumor growth through recruitment and activation of different subsets of cancer-associated fibroblasts. In seeking the molecular identity of the supporting factors provided by cancer-associated fibroblasts, we made use of antibody arrays and an in vivo coinjection model to identify osteopontin as the effector of the augmented tumor growth induced by PDGF-CC. In conclusion, we establish paracrine signaling by PDGF-CC as a potential drug target to reduce stromal support in malignant melanoma.