Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice
Moshe Elkabets, Ann M. Gifford, Christina Scheel, Bjorn Nilsson, Ferenc Reinhardt, Mark-Anthony Bray, Anne E. Carpenter, Karin Jirström, Kristina Magnusson, Benjamin L. Ebert, Fredrik Pontén, Robert A. Weinberg, Sandra S. McAllister
Moshe Elkabets, Ann M. Gifford, Christina Scheel, Bjorn Nilsson, Ferenc Reinhardt, Mark-Anthony Bray, Anne E. Carpenter, Karin Jirström, Kristina Magnusson, Benjamin L. Ebert, Fredrik Pontén, Robert A. Weinberg, Sandra S. McAllister
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Research Article

Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice

Abstract

Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+cKit– hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+cKit– BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.

Authors

Moshe Elkabets, Ann M. Gifford, Christina Scheel, Bjorn Nilsson, Ferenc Reinhardt, Mark-Anthony Bray, Anne E. Carpenter, Karin Jirström, Kristina Magnusson, Benjamin L. Ebert, Fredrik Pontén, Robert A. Weinberg, Sandra S. McAllister

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Figure 3

Sca1+cKit– hematopoietic BMCs are activated by instigating tumors.

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Sca1+cKit– hematopoietic BMCs are activated by instigating tumors. (...
(A) FACS density plots representing the collection of the following BMC populations from instigating tumor-bearing mice: Sca1+cKit+ (population i), Sca1 depleted (population ii), and Sca1+cKit– (population iii). BMC population collected from control mice: Sca1+cKit– (population iv). (B) Relative average mass of resulting tumors 12 weeks after subcutaneous injection of responder-BMC mixtures. Tumor mass was normalized to the mass of control responder tumors that had grown on their own (3 separate experiments; n = 18 per group). (C) Histopathology of responding tumors resulting from the indicated admixtures. Top panel shows αSMA staining of myofibroblasts and pericytes (brown) and hematoxylin counterstaining of nuclei (blue). Bottom panel shows Masson’s trichrome staining for collagen (blue) and cell nuclei (dark pink). Scale bar: 100 μm. (D) Representation of Sca1+cKit– BMCs as percentage of total BMCs from mice bearing Matrigel plugs (gray), noninstigating tumors (blue), or instigating tumors (red). (E) Flow cytometric analysis of Sca1+cKit– BMCs from mice bearing Matrigel plugs (gray), noninstigating tumors (blue), or instigating tumors (red). Histograms represent staining intensity for indicating cell-surface antigen markers. Graph represents average percentage of Sca1+cKit– cells that were positive for the indicated cell-surface antigens (n = 4 per group). No significant differences were observed between groups. (F) Partial heat map showing differential gene expression analysis of Sca1+cKit– BMCs from instigator-bearing mice (BPLER, n = 4) compared with those from size-matched noninstigator-bearing mice (PC3, n = 5). (G) Fold change of GRN mRNA expression (qPCR) in sorted Sca1+cKit– BMCs prepared from indicated mice (n = 4 per group). Data are expressed as mean ± SEM.