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STAT3 has important functions in both tumor cells and the tumor microenvironment to facilitate cancer progression. The STAT regulatory kinase Janus-activated kinase (JAK) has been strongly implicated in promoting oncogenesis of various solid tumors, including the use of JAK kinase inhibitors such as AZD1480. However, direct evidence that JAK drives STAT3 function and cancer pathogenesis at the level of the tumor microenvironment is yet to be established clearly. In this study, we show that AZD1480 inhibits STAT3 in tumor-associated myeloid cells, reducing their number and inhibiting tumor metastasis. Myeloid cell–mediated angiogenesis was also diminished by AZD1480, with additional direct inhibition of endothelial cell function in vitro and in vivo. AZD1480 blocked lung infiltration of myeloid cells and formation of pulmonary metastases in both mouse syngeneic experimental and spontaneous metastatic models. Furthermore, AZD1480 reduced angiogenesis and metastasis in a human xenograft tumor model. Although the effects of AZD1480 on the tumor microenvironment were important for the observed antiangiogenic activity, constitutive activation of STAT3 in tumor cells themselves could block these antiangiogenic effects, showing the complexity of the JAK/STAT signaling network in tumor progression. Together, our results indicated that AZD1480 can effectively inhibit tumor angiogenesis and metastasis mediated by STAT3 in stromal cells as well as tumor cells. Cancer Res; 71(21); 6601–10. ©2011 AACR.

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Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy., Understanding the tumor microenviroment is important before it can be exploited therapeutically. Here, the authors use single cell sequencing to study stromal cells in mouse tumors and identify a subset of interferon-licensed cancer associated fibroblasts that appear after anti-TGFβ treatment.

STAT3 has important functions in both tumor cells and the tumor microenvironment to facilitate cancer progression. The STAT regulatory kinase Janus-activated kinase (JAK) has been strongly implicated in promoting oncogenesis of various solid tumors, including the use of JAK kinase inhibitors such as AZD1480. However, direct evidence that JAK drives STAT3 function and cancer pathogenesis at the level of the tumor microenvironment is yet to be established clearly. In this study, we show that AZD1480 inhibits STAT3 in tumor-associated myeloid cells, reducing their number and inhibiting tumor metastasis. Myeloid cell–mediated angiogenesis was also diminished by AZD1480, with additional direct inhibition of endothelial cell function in vitro and in vivo. AZD1480 blocked lung infiltration of myeloid cells and formation of pulmonary metastases in both mouse syngeneic experimental and spontaneous metastatic models. Furthermore, AZD1480 reduced angiogenesis and metastasis in a human xenograft tumor model. Although the effects of AZD1480 on the tumor microenvironment were important for the observed antiangiogenic activity, constitutive activation of STAT3 in tumor cells themselves could block these antiangiogenic effects, showing the complexity of the JAK/STAT signaling network in tumor progression. Together, our results indicated that AZD1480 can effectively inhibit tumor angiogenesis and metastasis mediated by STAT3 in stromal cells as well as tumor cells. Cancer Res; 71(21); 6601–10. ©2011 AACR.

SummaryPersistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.

27 Background: The immune response to cancer is shaped by the abundance and localization of immune cell populations, their activation status, and expression of immunomodulatory factors. To detect proteins at high multiplex with spatial resolution, NanoString optical barcoding technology was used to digitally profile protein expression in formalin fixed paraffin embedded (FFPE) samples and compared to traditional IHC. Methods: NanoString has enabled digital profiling of immuno-oncology protein targets, including immune cell markers and checkpoint proteins. Protein detection is enabled via primary antibodies (Abs) which are attached via a UV cleavable linker to DNA indexing oligos. FFPE samples are stained with a multiplex cocktail of labeled Abs, then DNA oligos are released by UV light exposure. Liberated oligos are then hybridized to optical barcodes for quantitation on a NanoString instrument. This technique enables quantitative, multiplex protein detection over 5 logs of dynamic range. IHC and NanoString spatial protein profiling were performed on alternating sections from blocks of tonsil, melanoma, and colorectal cancer. Sections were fluorescently labeled with panCK, Ki67, and Syto-83 to visualize morphology, and regions of interest (ROI) were selected for profiling using up to 30 oligo-tagged Abs. Results: NanoString counts strongly correlated with quantification of CD3, CD4, CD8, PanCK, Ki67, PD-1, and PD-L1 derived from IHC. To explore limits of detection, ROIs of 1, 2, 4, or 8 cells were profiled. Digital counts were detected above background at the single cell level and linearly correlated with cell count. Furthermore, quantification with two cell lineage markers and double positive cells were demonstrated using a 30-plex Ab cocktail. Conclusions: Multiplex, digital protein profiling with spatial resolution will enable deep characterization of immune responses in tumors. Additionally, single cell profiling enables inter-cellular characterization of variations in immune response. The strong correlation of NanoString data to IHC indicates the feasibility to spatially profile multiple key proteins with minimal consumption of patient tissue.