Nathaniel D. Kirkpatrick

Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO(3) selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by (31)P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO(3) therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO(3) therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease.

CXCL12 (SDF1α)-CXCR4/CXCR7 Pathway Inhibition: An Emerging Sensitizer for Anticancer Therapies?

Addition of multiple molecularly targeted agents to the existing armamentarium of chemotherapeutics and radiotherapies represents a significant advance in the management of several advanced cancers. In certain tumor types with no efficacious therapy options, these agents have become the first line of therapy, for example, sorafenib in advanced hepatocellular carcinoma or bevacizumab in recurrent glioblastoma. Unfortunately, in many cases, the survival benefits are modest, lasting only weeks to a few months. Moreover, they may not show benefit in patients with localized disease (i.e., in the adjuvant setting). Recent studies have provided increasing evidence that activation of the chemokine CXCL12 (SDF1α) pathway is a potential mechanism of tumor resistance to both conventional therapies and biological agents via multiple complementary actions: (i) by directly promoting cancer cell survival, invasion, and the cancer stem and/or tumor-initiating cell phenotype; (ii) by recruiting “distal stroma” (i.e., myeloid bone marrow–derived cells) to indirectly facilitate tumor recurrence and metastasis; and (iii) by promoting angiogenesis directly or in a paracrine manner. Here, we discuss recent preclinical and clinical data that support the potential use of anti-CXCL12 agents (e.g., AMD3100, NOX-A12, or CCX2066) as sensitizers to currently available therapies by targeting the CXCL12/CXCR4 and CXCL12/CXCR7 pathways. Clin Cancer Res; 17(8); 2074–80. ©2011 AACR.

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

Endogenous Optical Biomarkers of Ovarian Cancer Evaluated with Multiphoton Microscopy

Purpose: Among gynecologic cancers, ovarian cancer is the second most common and has the highest mortality. Currently, there is no accurate early diagnostic technique for ovarian cancer. Furthermore, little is understood regarding the early progression of this disease. We have imaged multiphoton interactions of endogenous tissue constituents from normal and abnormal ovarian biopsies that were kept viable during transport from the operating room and microscopy. Experimental Design: The ovarian surface and underlying stroma were assessed with two-photon excited fluorescence (2PEF) and second harmonic generation (SHG). High-resolution, optically sectioned images were analyzed for epithelial morphology based on 2PEF and collagen density and structural integrity based on SHG. Additionally, multiwavelength 2PEF provided an estimation of the cellular redox ratio of epithelial cells. Results: Normal tissue exhibited a uniform epithelial layer with highly structured collagen in the stroma, whereas abnormal tissue exhibited varied epithelium with large cells and substantial quantitative changes to the collagen structure. Samples from patients at high risk for developing ovarian cancer (based on their personal/family history of cancer) exhibited highly variable cellular redox ratios and changes in collagen structure that trended toward cancer samples. Conclusion: This study highlights differences in endogenous signals in viable ovarian biopsies based on quantitative collagen structural changes and redox ratio estimates that may lead to improved detection and further insights in ovarian cancer, particularly in the early stages of the disease. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2048–55)

Endogenous fluorescence spectroscopy of cell suspensions for chemopreventive drug monitoring

Abstract Cancer chemopreventive agents such as N-4-(hydroxyphenyl)retinamide (4HPR) are thought to prevent cancers by suppressing growth or inducing apoptosis in precancerous cells. Mechanisms by which these drugs affect cells are often not known, and the means to monitor their effects is not available. In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment with 4HPR in ovarian and bladder cancer cell lines. Fluorescence signals consistent with nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan were measured to monitor cellular activity through redox status and protein content. Cells were treated with varying concentrations of 4HPR and measured in a stable environment with a sensitive fluorescence spectrometer. Results suggest that redox signal of all cells changed in a similar dose-dependant manner but started at different baseline levels. Redox signal changes depended primarily on changes consistent with NADH fluorescence, whereas the FAD fluorescence remained relatively constant. Similarly, tryptophan fluorescence decreased with increased drug treatment, suggesting a decrease in protein production. Given that each cell line has been shown to have a different apoptotic response to 4HPR, fluorescence redox values along with changes in tryptophan fluorescence may be a response as well as an endpoint marker for chemopreventive drugs.

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Angiopoietin-2 interferes with anti-VEGFR2- induced vessel normalization and survival benefit in mice bearing gliomas

Purpose: In brain tumors, cerebral edema is a significant source of morbidity and mortality. Recent studies have shown that inhibition of vascular endothelial growth factor (VEGF) signaling induces transient vascular normalization and reduces cerebral edema, resulting in a modest survival benefit in glioblastoma patients. During anti-VEGF treatment, circulating levels of angiopoietin (Ang)-2 remained high after an initial minor reduction. It is not known, however, whether Ang-2 can modulate anti-VEGF treatment of glioblastoma. Here, we used an orthotopic glioma model to test the hypothesis that Ang-2 is an additional target for improving the efficacy of current anti-VEGF therapies in glioma patients. Experimental Design: To recapitulate high levels of Ang-2 in glioblastoma patients during anti-VEGF treatment, Ang-2 was ectopically expressed in U87 glioma cells. Animal survival and tumor growth were assessed to determine the effects of Ang-2 and anti–VEGF receptor 2 (VEGFR2) treatment. We also monitored morphologic and functional vascular changes using multiphoton laser scanning microscopy and immunohistochemistry. Results: Ectopic expression of Ang-2 had no effect on vascular permeability, tumor growth, or survival, although it resulted in higher vascular density, with dilated vessels and reduced mural cell coverage. On the other hand, when combined with anti-VEGFR2 treatment, Ang-2 destabilized vessels without affecting vessel regression and compromised the survival benefit of VEGFR2 inhibition by increasing vascular permeability. VEGFR2 inhibition normalized tumor vasculature whereas ectopic expression of Ang-2 diminished the beneficial effects of VEGFR2 blockade by inhibiting vessel normalization. Conclusion: Cancer treatment regimens combining anti-VEGF and anti-Ang-2 agents may be an effective strategy to improve the efficacy of current anti-VEGF therapies. Clin Cancer Res; 16(14); 3618–27. ©2010 AACR.

Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma

Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas, but tumors invariably recur. Because tumor-associated macrophages have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of macrophages in patients with recurrent glioblastoma. We compared autopsy brain specimens from 20 patients with recurrent glioblastoma who received antiangiogenic treatment and chemoradiation with 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy or no treatment. Tumor-associated macrophages were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in macrophages in the antiangiogenic-treated patients. Immunohistochemical analysis demonstrated an increase in CD68+ macrophages in the tumor bulk (P < .01) and infiltrative areas (P = .02) in antiangiogenic-treated patients. We also observed an increase in CD11b+ cells in the tumor bulk (P < .01) and an increase in CD163+ macrophages in infiltrative tumor (P = .02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumors (P = .05 and P = .05, respectively) correlated with poor overall survival among patients who first received antiangiogenic therapy at recurrence. In summary, recurrent glioblastomas showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival among these patients. These data suggest that tumor-associated macrophages may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in recurrent glioblastoma.

Cancer cell associated MT1-MMP promotes blood vessel invasion and distant metastasis in triple-negative mammary tumors

Functional roles for the cancer cell-associated membrane type I matrix metalloproteinase (MT1-MMP) during early steps of the metastatic cascade in primary tumors remain unresolved. In an effort to determine its significance, we determined the in vivo effects of RNAi-mediated downregulation in mammary cancer cells on the migration, blood and lymphatic vessel invasion (LVI), and lymph node and lung metastasis. We also correlated the expression of cancer cell MT1-MMP with blood vessel invasion (BVI) in 102 breast cancer biopsies. MT1-MMP downregulation in cancer cells decreased lung metastasis without affecting primary tumor growth. The inhibition of lung metastasis correlated with reduced cancer cell migration and BVI. Furthermore, cancer cell-expressed MT1-MMP upregulated the expression of MT1-MMP in vascular endothelial cells, but did not affect MT1-MMP expression in lymphatic endothelial cells, LVI, or lymph node metastasis. Of clinical importance, we observed that elevated MT1-MMP expression correlated with BVI in biopsies from triple-negative breast cancers (TNBC), which have a poor prognosis and high incidence of distant metastasis, relative to other breast cancer subtypes. Together, our findings established that MT1-MMP activity in breast tumors is essential for BVI, but not LVI, and that MT1-MMP should be further explored as a predictor and therapeutic target of hematogenous metastasis in TNBC patients.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Significance Inhibition of the VEGF/VEGF receptor (VEGFR) pathway has failed to increase overall survival in phase III trials in patients with glioblastoma (GBM). Previously we identified the angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway as a potential driver of resistance to VEGF inhibition in GBM. Here we show that dual inhibition of VEGFRs and Ang-2 inhibits tumor growth and prolongs vessel normalization compared with VEGFR inhibition alone, resulting in improved survival in murine GBM models. Furthermore, by blocking macrophage recruitment, we demonstrate that macrophages contribute to the beneficial effects of dual therapy. Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.