Elizabeth S. Nakasone

Tumors as organs: complex tissues that interface with the entire organism

Solid tumors are not simply clones of cancer cells. Instead, they are abnormal organs composed of multiple cell types and extracellular matrix. Some aspects of tumor development resemble processes seen in developing organs, whereas others are more akin to tissue remodeling. Some microenvironments, particularly those associated with tissue injury, are favorable for progression of mutant cells, whereas others restrict it. Cancer cells can also instruct surrounding tissues to undergo changes that promote malignancy. Understanding the complex ways in which cancer cells interact with their surroundings, both locally in the tumor organ and systemically in the body as a whole, has implications for effective cancer prevention and therapy.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

Treatment with DNase I–coated nanoparticles prevents metastasis by targeting neutrophil extracellular traps induced by cancer cells in a mouse model. Metastasis caught in a NET Neutrophil extracellular traps, or NETs, are DNA structures that are produced by neutrophils in response to infection and can promote the spread of cancer in the presence of infection. Park et al. discovered that even in the absence of infection, metastatic breast cancer cells can stimulate neutrophils to form NETs, which further support the spread of metastasis. The authors also demonstrated an approach to breaking this vicious cycle using nanoparticles coated with DNase I, an enzyme that breaks down DNA NETs. This treatment was effective in reducing lung metastases in mice, demonstrating the potential of NETs as a therapeutic target. 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. 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 deoxyribonuclease I (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.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

Abstract IA11: Understanding drug responses and resistance mechanisms using imaging in live mice

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.

Abstract A11: Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis

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.