The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.

High serum levels of IL-6 correlate with poor outcome in breast cancer patients. However, no data are available on the relationship between IL-6 and mammary stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in the MCF-7 breast cancer cell line and in primary human mammospheres (MS), multicellular structures enriched in stem/progenitor cells of the mammary gland. MS from node invasive breast carcinoma tissues expressed IL-6 mRNA at higher levels than did MS from matched nonneoplastic mammary glands. In addition, IL-6 mRNA was detected only in basal-like breast carcinoma tissues, an aggressive breast carcinoma variant showing stem cell features. IL-6 treatment triggered Notch-3-dependent upregulation of the Notch ligand Jagged-1 and promotion of MS and MCF-7-derived spheroid growth. Moreover, IL-6 induced Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promoted a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, autocrine IL-6 signaling relied upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, these data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

SignificanceIncreasing evidence suggests that extracellular vesicles (EVs) can transfer genetic material to recipient cells. However, the mechanism and role of this phenomenon are largely unknown. Here we have made a remarkable discovery: EVs can harbor the full mitochondrial genome. These extracellular vesicles can in turn transfer their mtDNA to cells with impaired metabolism, leading to restoration of metabolic activity. We determined that hormonal therapy induces oxidative phosphorylation-deficient breast cancer cells, which can be rescued via the transfer of mtDNA-laden extracellular vesicles. Horizontal transfer of mtDNA occurred in cancer stem-like cells and was associated with increased self-renewal potential of these cells, leading to resistance to hormonal therapy. We propose that mtDNA transfer occurs in human cancer via EVs.

The Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway was discovered 20 years ago as a mediator of cytokine signaling. Since this time, more than 2,500 articles have been published demonstrating the importance of this pathway in virtually all malignancies. Although there are dozens of cytokines and cytokine receptors, four Jaks, and seven Stats, it seems that interleukin-6-mediated activation of Stat3 is a principal pathway implicated in promoting tumorigenesis. This transcription factor regulates the expression of numerous critical mediators of tumor formation and metastatic progression. This review will examine the relative importance and function of this pathway in nonmalignant conditions as well as malignancies (including tumor intrinsic and extrinsic), the influence of other Stats, the development of inhibitors to this pathway, and the potential role of inhibitors in controlling or eradicating cancers.

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

Common sites of breast cancer metastasis include the lung, liver, and bone, and of these secondary metastatic sites, estrogen receptor A (ERA)-positive breast cancer often favors bone. Within secondary organs, cancer cells would predictably encounter tissue-specific fibroblasts or their soluble factors, yet our understanding of how tissue-specific fibroblasts directly affect cancer cell growth rates and survival remains largely unknown. Therefore, we tested the hypothesis that mesenchymal fibroblasts isolated from common sites of breast cancer metastasis provide a more favorable microenvironment with respect to tumor growth rates. We found a direct correlation between the ability of breast, lung, and bone fibroblasts to enhance ERA-positive breast cancer cell growth and the level of soluble interleukin-6 (IL-6) produced by each organ-specific fibroblast, and fibroblast-mediated growth enhancement was inhibited by the removal or inhibition of IL-6. Interestingly, mice coinjected with MCF-7 breast tumor cells and senescent skin fibroblasts, which secrete IL-6, developed tumors, whereas mice coinjected with presenescent skin fibroblasts that produce little to no IL-6 failed to form xenograft tumors. We subsequently determined that IL-6 promoted growth and invasion of breast cancer cells through signal transducer and activator of transcription 3-dependent up-regulation of Notch-3, Jagged-1, and carbonic anhydrase IX. These data suggest that tissue-specific fibroblasts and the factors they produce can promote breast cancer disease progression and may represent attractive targets for development of new therapeutics. [Cancer Res 2008;68(21):9087-95]

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The comprehension of the basic biology of stem cells is expected to provide a useful insight into the pathogenesis of cancer. In particular, there is evidence that hypoxia promotes stem cell renewal in vitro as well as in vivo. It therefore seems reasonable that stem cell survival and hypoxia response are strictly connected at molecular level. We here report that the 66-kDa isoform of the SHC gene (p66Shc) is induced in a breast cancer cell line by the exposure to hypoxic environment and that it controls the expression of the stem cell regulatory gene Notch-3. Then, we show that p66Shc/Notch-3 interplay modulates self-renewal (by inducing the Notch-ligand Jagged-1) and hypoxia survival (by inducing the hypoxia-survival gene carbonic anhydrase IX) in mammary gland stem/progenitor cells, expanded in vitro as multicellular spheroids (mammospheres). We conclude that mechanisms that regulate stem cell renewal and hypoxia survival are integrated at the level of the p66Shc/Notch3 interplay. Because Notch-3, Jagged-1, and carbonic anhydrase IX are dysregulated in breast cancer, and because p66Shc is an aging-regulating gene, we envision that these data may help in understanding the relationship among aging, cancer, and stem cells. STEM CELLS 2007;25:807-815