Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. Here, we show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with DNase I blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

SUMMARY Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses in situ.

It is well appreciated that factors intrinsic to the cancer cells, such as specific mutations, regulate response to chemotherapy. However, there is limited knowledge on the dynamics of cancer cell death in response to therapy in the naturally developing tumor microenvironment. Using intravital microscopy (microscopy in live mice) of tumors, we show that factors within the microenvironment, extrinsic to the cancer cells, support the development of chemoresistance by regulating drug distribution and the inflammatory response. Specifically, intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to the chemotherapeutic drug doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. These results have clinical implications, as myeloid cell infiltration is increased in human breast tumors after chemotherapy and the cellular composition of the immune infiltrate is a strong predictor of overall survival. Our data further suggest that the response to classical chemotherapeutic drugs can be improved by changing the tumor microenvironment with agents that modify matrix metalloproteinase activity and chemokine signaling. Thus, intravital imaging can be used to gain insights into drug responses in situ. Citation Format: Mikala Egeblad, Elizabeth Nakasone, Hanne Askautrud, Robert Wysocki, Miriam Fein, Tim Kees, Juwon Park, Jae-Hyun Park. Understanding drug responses and resistance mechanisms using imaging in live mice. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA11.

Innate immune cells such as macrophages, monocytes and neutrophils can be recruited to tumors by chemokines, which act on chemokines receptors on these cells. Macrophages and monocytes have well-established roles in promotion of metastasis. However, neutrophils have been reported to have both pro- and anti-metastatic effects. In this study, we used the metastatic 4T1 and non-metastatic 4T07 murine breast carcinoma cells lines (originating from the same tumor) to study effects of neutrophils in breast metastasis. Primary tumors from 4T1 cells, orthotopically injected into syngeneic mice, had more infiltrating neutrophils than did primary tumors from 4T07 cells. The increased numbers of neutrophils, which expressed the chemokine receptor CXCR2, was associated with significantly higher levels of cancer cell-secreted CXCL1, a chemokine ligand for CXCR2. To test whether cancer cell-secreted CXCL1 acted on neutrophils to regulate metastasis, we used shRNAs to reduce CXCL1 expression in the 4T1 cells. Consistent with the lack of CXCR2 expression by the cancer cells, this did not influence cancer cell proliferation or migration in vitro. However, in vivo, tumor infiltration of neutrophils and lung metastasis were both reduced. Moreover, when parental 4T1 cells were orthotopically injected into syngeneic Cxcr2-/-mice, neutrophil infiltration and lung metastasis were significantly decreased as compared to tumors in wild type littermate. These results suggest that paracrine CXCL1 by cancer cells facilitates neutrophil infiltration via CXCR2 and that neutrophils in turn promote lung metastasis. To examine the mechanisms by which interactions between cancer cells and neutrophils might promote lung metastasis, we performed co-culture experiments with cancer cells and neutrophils. We found that neutrophils directly stimulated 4T1 cell invasion through matrigel. Interestingly, cancer-released CXCL1 also promoted the formation of neutrophil extraceullar traps (NETs), which is an expulsion of DNA and proteases from the neutrophils. Both invasion and NET formation were blocked by inhibition of phagocyte NADPH oxidase. Moreover, when mice with 4T1 tumors were treated with a phagocyte NADPH oxidase inhibitor, tumor growth and lung metastasis was reduced. Finally, decreased levels of the phosphorylated form of p47phox, a cytosolic component of phagocyte NAPDH oxidase that is activated by phosphorylation, was decreased in the tumor lysates from 4T1 injected Cxcr2-/- mice as compared to Cxcr2+/- mice. In conclusion, our study revealed that cancer cell-secreted CXCL1 regulates neutrophil infiltration and extracellular trap formation, and that phagocyte NADPH oxidase activity in these neutrophils promoted cancer cell invasion and metastasis. Citation Format: Juwon Park, Jing Qiu, Elizabeth S. Nakasone, Mikala Egeblad. Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A11.

The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of multiple myeloma cells. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support multiple myeloma cell survival. In this study, we found that CD44s and CD44 variants were differentially expressed between fraction of CD138+ fraction and CD138- fraction. Expression levels of CD44v6, CD44v9, and CD44v10, respectively, correlated with bone erosion (p=0.029, p=0.013, p=0.032), suggesting that CD44 variant molecules are involved in multiple myeloma progression. Binding studies using CD44 isoform specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM. In 3D culture, CD44v6 and CD44v9-mediated plasma cell binding resulted in a significant induction of HGF secretion by bone marrow stromal cells. CD44v6 and CD44v9-mediated plasma cell binding significantly induces PI3K/Akt via activation the Src-kinase Lyn. In bone marrow serum of MM patients, the expression levels of IL-6, OPN, and hepatocyte growth factor (HGF), respectively, statistically correlated with bone erosion of MM patients (p=0.021, p=0.001, p=0.036). HGF derived from bone marrow stromal cells with multiple myeloma cells stimulates CD44 signaling via activation of HGF-receptor/cMet. Specific CD44 shRNA suppresses HGF-mediated CD44 signaling. Taken together, the role of CD44 variants in adhesion induced HGF- secretion may explain the previously observed correlation between CD44 variants expression and adverse prognosis in multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1527. doi:1538-7445.AM2012-1527