Dorothy A. Sipkins

In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.

The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.

Leukemic Cells Create Bone Marrow Niches That Disrupt the Behavior of Normal Hematopoietic Progenitor Cells

The host tissue microenvironment influences malignant cell proliferation and metastasis, but little is known about how tumor-induced changes in the microenvironment affect benign cellular ecosystems. Applying dynamic in vivo imaging to a mouse model, we show that leukemic cell growth disrupts normal hematopoietic progenitor cell (HPC) bone marrow niches and creates abnormal microenvironments that sequester transplanted human CD34+ (HPC-enriched) cells. CD34+ cells in leukemic mice declined in number over time and failed to mobilize into the peripheral circulation in response to cytokine stimulation. Neutralization of stem cell factor (SCF) secreted by leukemic cells inhibited CD34+ cell migration into malignant niches, normalized CD34+ cell numbers, and restored CD34+ cell mobilization in leukemic mice. These data suggest that the tumor microenvironment causes HPC dysfunction by usurping normal HPC niches and that therapeutic inhibition of HPC interaction with tumor niches may help maintain normal progenitor cell function in the setting of malignancy.

ICAM-1 expression in autoimmune encephalitis visualized using magnetic resonance imaging

The expression of leukocyte adhesion molecules in the intact brains of mice with experimental autoimmune encephalitis (EAE) was visualized by Magnetic Resonance Imaging (MRI) through the use of a new, target-specific MR contrast agent. Antibody-conjugated paramagnetic liposomes (ACPLs) were designed to achieve in vivo targeting of molecules expressed on vascular endothelium, while providing sufficient signal enhancement at these sites for detection by MRI. ACPLs targeted to intercellular adhesion molecule-1 (ICAM-1), an endothelial leukocyte receptor upregulated on cerebral microvasculature during EAE, were administered to diseased mice. Fluorescence microscopy confirmed that fluorescently-tagged ACPLs were localized to central nervous system (CNS) microvasculature in a pattern consistent with ICAM-1 upregulation described immunohistochemically. High resolution MRI of mouse brains ex vivo demonstrated that ACPL binding conferred significant enhancement of signal intensity (SI) as compared to control images. These results suggest that ACPLs can be used as MRI contrast agents to visualize specific molecules expressed on vascular endothelium during disease.

Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy

Malignant cells may evade death from cytotoxic agents if they are in a dormant state. The host microenvironment plays important roles in cancer progression, but how niches might control cancer cell dormancy is little understood. Here we show that osteopontin (OPN), an extracellular matrix molecule secreted by osteoblasts, can function to anchor leukemic blasts in anatomic locations supporting tumor dormancy. We demonstrate that acute lymphoblastic leukemia (ALL) cells specifically adhere to OPN in vitro and secrete OPN when localized to the endosteal niche in vivo. Using intravital microscopy to perform imaging studies of the calvarial bone marrow (BM) of xenografted mice, we show that OPN is highly expressed adjacent to dormant tumor cells within the marrow. Inhibition of the OPN-signaling axis significantly increases the leukemic cell Ki-67 proliferative index and leads to a twofold increase in tumor burden in treated mice. Moreover, using cell-cycle-dependent Ara-C chemotherapy to produce minimal residual disease (MRD) in leukemic mice, we show that OPN neutralization synergizes with Ara-C to reduce detectable BM MRD. Taken together, these data suggest that ALL interacts with extracellular OPN within the malignant BM, and that this interaction induces cell cycle exit in leukemic blasts, protecting them from cytotoxic chemotherapy.

The stem cell niche in health and malignancy

Somatic stem cells play a well-defined and important role in tissue renewal. Their malignant counterparts, cancer stem cells, are thought to be responsible for tumor initiation and possibly chemotherapy resistance, although controversy remains regarding both the origin and characterization of these cells. Both somatic and cancer stem cells appear to occupy specialized microenvironments in many organs. These niches are important for both maintenance of quiescence and control of cellular survival and proliferation. Targeting cancer stem cells and their microenvironments may provide new therapies to eradicate tumors. The efficacy of several drugs in current use is mediated at least in part via effects on the microenvironment, and new drugs that target the niche are currently in clinical trials.

Dormant breast cancer micrometastases reside in specific bone marrow niches that regulate their transit to and from bone

Breast cancer cells traffic to and from the peripheral blood within specific vascular niches, and this migration can be therapeutically targeted. Taking away cancer’s hideouts Breast cancer is notorious for its ability to relapse after many years, long after a patient had completed treatment. Price et al. demonstrate that the culprits responsible for such late metastasis may be dormant cancer cells hiding in perivascular niches. The authors showed that proteins called E-selectin and CXCR4 exert different forces on these cancer cells, with CXCR4 anchoring breast cancer cells to their niches and E-selectin allowing entry of cancer cells into the bone marrow. These findings suggest that combining a CXCR4 inhibitor to force the cells out of their niches and an E-selectin inhibitor to prevent metastasis to the bone marrow could help trap the cells in the vasculature, where they could be killed with chemotherapy. Breast cancer metastatic relapse can occur years after therapy, indicating that disseminated breast cancer cells (BCCs) have a prolonged dormant phase before becoming proliferative. A major site of disease dissemination and relapse is bone, although the critical signals that allow circulating BCCs to identify bone microvasculature, enter tissue, and tether to the microenvironment are poorly understood. Using real-time in vivo microscopy of bone marrow (BM) in a breast cancer xenograft model, we show that dormant and proliferating BCCs occupy distinct areas, with dormant BCCs predominantly found in E-selectin– and stromal cell–derived factor 1 (SDF-1)–rich perisinusoidal vascular regions. We use highly specific inhibitors of E-selectin and C-X-C chemokine receptor type 4 (CXCR4) (SDF-1 receptor) to demonstrate that E-selectin and SDF-1 orchestrate opposing roles in BCC trafficking. Whereas E-selectin interactions are critical for allowing BCC entry into the BM, the SDF-1/CXCR4 interaction anchors BCCs to the microenvironment, and its inhibition induces mobilization of dormant micrometastases into circulation. Homing studies with primary BCCs also demonstrate that E-selectin regulates their entry into bone through the sinusoidal niche, and immunohistochemical staining of patient BMs shows dormant micrometastatic disease adjacent to SDF-1+ vasculature. These findings shed light on how BCCs traffic within the host, and suggest that simultaneous blockade of CXCR4 and E-selectin in patients could molecularly excise dormant micrometastases from the protective BM environment, preventing their emergence as relapsed disease.

Real-time Detection of Circulating Apoptotic Cells by In Vivo Flow Cytometry

Survival of cancer cells in the circulation is an important step in metastasis. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the first in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method [1–3] that enables real-time detection and quantification of circulating cells without blood extraction. Injected cancer cells undergo cell death within 1–2 hr after entering the mouse circulation. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 min. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention. To detect circulating apoptotic cells in vivo, annexin-V conjugated to Alexa Fluor 647 (AF647) was used to label exposed phosphatidylserine on the cell surface. The long wavelength of AF647 fluorescence (Molecular Probes, OR) allows its detection through blood with minimum attenuation by red blood cells. In initial studies conducted to demonstrate that our instrument had sufficient sensitivity to detect individual annexin-Vlabeled apoptotic cells in vivo, we used MatLyLu prostate cancer cells pretreated with camptothecin [4] as a positive control. We verified that >80% of the camptothecin-treated cells undergo apoptosis by conventional flow cytometry (i.e., >80% of the treated cells were FITC–annexin V positive and propidium iodide negative). The camptothecin-treated cells were then labeled with the AF647-conjugated annexin-V and injected into the mouse intravenously. The circulating annexin-V + cells were measured by focusing a He–Ne laser beam onto an ear vessel and detecting the fluorescent bursts as individual cells flowed

A novel clofarabine bridge strategy facilitates allogeneic transplantation in patients with relapsed/refractory leukemia and high-risk myelodysplastic syndromes

Patients with relapsed/refractory leukemias or advanced myelodysplastic syndrome (MDS) fare poorly following allogeneic hematopoietic cell transplant (HCT). We report prospective phase II study results of 29 patients given clofarabine 30 mg/m2/day i.v. × 5 days followed immediately by HCT conditioning while at the cytopenic nadir. A total of 15/29 patients (52%) were cytoreduced according to pre-defined criteria (cellularity <20% and blasts <10%). Marrow cellularity (P<0.0001) and blast% (P=0.03) were reduced. Toxicities were acceptable, with transient hyperbilirubinemia (48%) and gr3–4 infections (10%). In all, 28/29 proceeded to transplant; 27 received ATG or alemtuzumab. Post HCT, 180 day non-relapse mortality (NRM) was 7% (95% confidence interval (CI): 1–21), relapse was 29% (95% CI: 13–46) and OS was 71% (95% CI: 51–85), comparing favorably to published data for high-risk patients. Two-year graft vs host disease incidence was 40% (95% CI: 21–58) and 2 year OS was 31% (95% CI: 14–48). Disease at the nadir correlated with inferior OS after HCT (HR=1.22 for each 10% marrow blasts, 95% CI: 1.02–1.46). For AML/MDS patients, there was a suggestion that successful cytoreduction increased PFS (330 vs 171 days, P=0.3) and OS (375 vs 195 days, P=0.31). Clofarabine used as a bridge to HCT reduces disease burden, is well tolerated, and permits high-risk patients to undergo HCT with acceptable NRM. Late relapses are common; thus, additional strategies should be pursued. NCT-00724009.

High dose cytarabine and mitoxantrone: an effective induction regimen for high-risk Acute Myeloid Leukemia (AML)

Abstract Patients with high-risk AML, defined as those with advanced age, relapsed/refractory disease, unfavorable molecular and cytogenetic abnormalities, therapy-related myeloid neoplasm (t-MN) and multiple medical co-morbidities tend to respond poorly to standard cytarabine and daunorubicin induction therapy and have a poor prognosis. We performed a retrospective analysis of an alternative induction regimen using high dose cytarabine (HiDAC) and mitoxantrone (MITO) administered to 78 high-risk patients with AML at The University of Chicago from 2001 to 2008. The primary endpoints of the study were complete remission (CR) rate and death within 30 days of initiation of treatment. The median age was 63 years (range:23–85); 27% of these patients had a Charlson co-morbidity index (CCI) > 2. Forty-three (56%) patients had unfavorable cytogenetics, 28 (37%) had intermediate-risk cytogenetics and 5 (7%) had favorable cytogenetics. The CR rate was 45% and the CRi rate 10%; 7 patients (9%) died during induction. Notably, t-MN and relapsed/refractory patients had CR and induction death rates equivalent to de novo AML patients within this series. In this high risk AML population, HiDAC/MITO induction demonstrated an overall response rate of 55% with a low induction death rate of 9% and allowed 32 (41%) patients to proceed to allogeneic stem cell transplant.

Rendering the Leukemia Cell Susceptible to Attack

In mice, short-term treatment with interferon-α pushes dormant hematopoietic stem cells into a state of proliferation. This finding may be relevant to the treatment of patients with chronic myelogenous leukemia, and treatment may be more effective if patients are first “primed” with interferon-α.