Despite the recent advances in the treatment of blood cancers, the great majority of malignancies of the B-lymphocyte lineage, including CLL, NHL and MM, remain incurable. A key reason for the difficulties to treat B-lineage neoplasms more effectively is the support for tumor cells provided by the tumor microenvironment (TME). Overcoming TME-dependent tumor promotion and disease relapse is thus an important goal of therapy.1 The TME is also a prime target for new approaches to tumor prevention, because it has an important role in neoplastic B cell and plasma cell (PC) development.2 However, strategies to specifically target the TME are often complicated by uncertainty as to whether the tumor-promoting factor considered for molecular targeting is indeed produced by nonmalignant bystander cells, as opposed to tumor cells. In circumstances in which the factor may be generated by both sources, such as interleukin-6 (IL-6) in MM,3,4 we do not know whether the TME-derived portion is critical for myeloma cells and, therefore, worth exploring for new TME-targeted therapies.
We developed an experimental model system to address this issue, using adoptive B cell transfer as the principal research tool. To illustrate the utility of this new method, we evaluated the role of IL-6 in plasma cell tumor (PCT) development in mice. We chose IL-6 because it drives PC neoplasia in both humans and mice and can be produced by both tumor and stromal cells.5 Additionally, despite the early recognition of its critical importance for MM, the promise of IL-6 as a therapeutic target in myeloma has not yet been translated into tangible clinical benefits.6 Here we demonstrate that stromal cell-derived ‘paracrine’ IL-6 is critical for PCT, whereas B cell-derived ‘autocrine’ IL-6 is dispensable. This finding provides proof of principle that the complex pathophysiological interactions of malignant PCs and the TME can be genetically dissected by means of adoptive B cell transfer in mice.
To determine the biological significance of IL-6 for inflammation-dependent PCT in mice, IL-6-deficient (IL6−) Myc-transgenic (Myc+)7 mice (n = 6) were treated i.p. with pristane. IL-6-proficient (IL6+) Myc+ mice were included as controls (n = 41). Both groups of mice were monitored for PCT until they reached 220 days of age. Tumor development in Myc+IL6+ mice was fully penetrant (100% PCT incidence) and rapid (126 days median survival), similar to results from a previous study on PCT that relied on a different Myc transgene.8 In striking contrast, none of the Myc+IL6− mice developed PCT (Figure 1a). This finding extended published results on the resistance of IL-6-deficient BALB/c (C) mice to PCT9 in demonstrating that tumor resistance was maintained in the presence of the tumor-promoting Myc transgene. We conclude that IL-6 is essential for PCT development, even in the mice genetically engineered to rapidly undergo neoplastic PC development with complete penetrance.
To evaluate the relative importance of B cell-derived ‘autocrine’ IL-6 and TME-derived ‘paracrine’ IL-6 for PCT, it was necessary to develop an experimental model system in which either source of the cytokine could be genetically eliminated. To do this, we evaluated whether Myc+ B cells adoptively transferred to sublethally irradiated hosts would undergo neoplastic PC development. Myc+B220+CD45.2+ splenocytes were isolated at ≥95% purity (Supplementary Figure 1A) and transferred to whole body-irradiated CD45.2+ (n = 16) or CD45.1+ (n = 8) mice treated 1 week later with i.p. pristane to induce the granulomatous tissue wherein PCTs arise (Figure 1b, top). Tumor development was complete and equally rapid (123 days median onset) in both groups of B cell-reconstituted hosts (Figure 1b, center left). Invariably, the neoplasms were confined to the peritoneal cavity (Supplementary Figure 1B) and expressed CD138 (Figure 1b, center right). Flow cytometric analysis using specific antibodies to CD45.2 (donor) and CD45.1 (host) demonstrated that PCTs were of donor cell origin and consisted predominantly of mature CD138+B220−CD19− PCs (Figure 1b, bottom). B-lymphocytes, which are also present in tumor-bearing ascites, were of donor type (Supplementary Figure 1C). These results indicated that the ‘adoptive transfer’ model of PCT is as effective in terms of tumor induction as the parental model using Myc+IL6+ mice (Figure 1a).
To further assess the suitability of the adoptive transfer model for studies on PCT, we investigated the biological properties of Myc+ B cells in greater depth. Myc+ and normal B cells developed comparably in the bone marrow (Supplementary Figure 2A) but exhibited changes in the proportions of follicular, transitional and B1a B cells in the spleen (Supplementary Figure 2). Despite this developmental difference, Myc+ and normal B cells harbored identical levels of the IL-6 receptor (IL-6R) and were both capable of secreting IL-6 in response to stimulation (Supplementary Figure 3A). Myc+ and normal B cells engrafted similarly in the spleens of mice not treated with pristane (Supplementary Figure 3B) and failed to develop PCT during an observation period of 220 days (Supplementary Figure 3C). These results underlined the importance of peritoneal inflammation for tumor development (Supplementary Figure 4) and supported the use of Myc+ B cells for a comparison of the efficacy with which autocrine and paracrine IL-6 promote inflammation-dependent PCT.
Transfer of Myc+IL6− or Myc+IL6+ B cells to IL6+ hosts resulted in both cases in fully penetrant tumor development with median onsets of 131 days (n = 8) and 126 days (n = 14), respectively, (P = 0.976; Figure 2a). Microarray-based global gene expression profiling of tumors from adoptively transferred mice demonstrated (i) a close match with the gene expression program of PCT that arose spontaneously in double-transgenic IL6Myc mice7 (Figure 2b, left) and (b) greater pathway-based similarity with human myeloma than with other malignant or normal human B-lineage cells (Figure 2b, right). Transfer of Myc+IL6+ B cells to IL6− recipients (n = 10) was associated with a significant delay in tumor onset (203 days median) relative to IL6+ hosts from this study (131 days; P < 0.001) or the study presented in Figure 1b (123 days; P < 0.001). Preliminary comparisons of neoplasms from IL6− and IL6+ hosts demonstrated no differences in CD138 expression as assessed by flow cytometry, PAX5 expression determined by immunohistochemistry, and a range of histopathologic features observed on examination of H&E-stained tissue sections (Figure 2c). However, the tumors differed in IRF4 immunoreactivity patterns, which were dominated by nuclear and coarse cytoplasmic staining in cases from IL6− and IL6+ mice, respectively (Figure 2d). We conclude that TME-derived IL-6 is important for PCT development, whereas B cell-derived IL-6 is dispensable (Figure 2a). Paracrine sources of IL-6 may also be critical for tumor development in previously developed mouse models of human MM driven by a widely expressed IL6 transgene7 (Supplementary Figure 5).
The chief result of this study is genetic evidence that non-malignant bystander cells in the TME are the main source of IL-6 for PCT in mice.10 Although IL-6 production by tumor precursors is sufficient to drive PCT when TME-derived IL-6 is lacking, tumor development is significant delayed as it depends on alternative, less efficient mechanisms of malignant PC transformation. These may include IL-6R-independent pathways11 driven by IL-2112 and IL-6R-dependent pathways activated by IL-11, IL-27, IL-31 and oncostatin M.13 Considering that lack of paracrine IL-6 prolonged PCT onset by ~50%, it is likely that targeting IL-6 production in human bone marrow stromal cells would slow the transition from MGUS14 to frank MM with similar efficiency, potentially preventing many cases of newly diagnosed disease. The adoptive cell transfer approach described here can be readily extended to studies on other myeloma drivers that govern complex tumor-TME interactions, such as Bruton tyrosine kinase (BTK).15 For example, complementary transfers of Myc+BTK− B cells to BTK+ hosts or Myc+BTK+ B cells to BTK− hosts may further our understanding of the specific function of BTK in myeloma cells vs osteoclasts and, thereby, provide preclinical support for the clinical testing of small-compound BTK inhibitors in myeloma.
Supplementary Material
ACKNOWLEDGEMENTS
This research was performed by TRR in partial fulfilment of the requirements for the degree Doctor of Philosophy in the Graduate Immunology Program of the University of Iowa. We thank Kristin Ness for expert mouse husbandry. This work was supported in part by NIH Predoctoral Training Grant 5T32 AI007485 (TRR), by the Intramural Research Program of the NIAID (to HCM), by NCI Core Grant P30CA086862 in support of The University of Iowa Holden Comprehensive Cancer Center, by a Senior Research Award from the Multiple Myeloma Research Foundation (to SJ), by a research award from the International Waldenström’s Macroglobulinemia Foundation (to SJ), by a NCI P50CA097274 career development award (to SJ), and by R01CA151354 from the NCI (to SJ).
Footnotes
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
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