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[Cancer Research 63, 4342-4346, August 1, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Maximum Tolerable Dose and Low-Dose Metronomic Chemotherapy Have Opposite Effects on the Mobilization and Viability of Circulating Endothelial Progenitor Cells1

Francesco Bertolini2, Saki Paul, Patrizia Mancuso, Silvia Monestiroli, Alberto Gobbi, Yuval Shaked and Robert S. Kerbel

Division of Hematology-Oncology, Department of Medicine [F. B., S. P., P. M.] and Department of Experimental Oncology, IFOM-Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology [S. M., A. G.], European Institute of Oncology, 20141 Milan, Italy; Molecular and Cell Biology Research, Sunnybrook and Women’s College Health Sciences Centre, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4N 3M5 Canada [Y. S., R. S. K.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
There is growing evidence that vasculogenesis (progenitor cell-derived generation of new blood vessels) is required for the growth of some neoplastic diseases. Here we show that the administration of cyclophosphamide (CTX) at the maximum tolerable dose with 21-day breaks or at more frequent low-dose (metronomic) schedules have opposite effects on the mobilization and viability of circulating endothelial progenitors (CEPs) in immunodeficient mice bearing human lymphoma cells. Animals treated with the maximum tolerable dose CTX experienced a robust CEP mobilization a few days after the end of a cycle of drug administration, and tumors rapidly became drug resistant. Conversely, the administration of metronomic CTX was associated with a consistent decrease in CEP numbers and viability and with more durable inhibition of tumor growth. Our findings suggest that metronomic low-dose chemotherapy regimens are particularly promising for avoiding CEP mobilization and, hence, to potentially reduce vasculogenesis-dependent mechanisms of tumor growth.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
A number of recent experimental observations have suggested that the growth of some types of cancer may depend on vasculogenesis (i.e., progenitor-cell dependent generation of new blood vessels) and not just angiogenesis (i.e., mature endothelial-cell dependent generation of new blood vessels) alone. The presence of marrow-derived CEP3 able to participate in adult vasculogenesis was first demonstrated by Asahara et al. (1) in a murine model and by Shi et al. (2) in a canine bone marrow transplantation model in which a dacron graft was colonized by donor endothelial cells. Regarding neoplastic diseases, the first proof of principle of the pivotal role of CEP in tumor growth was recently obtained in the Id1+/- Id3-/- murine model (3) . In these mice, most tumors failed to grow progressively, if at all, and any tumor growth present remained at a quiescent state because of the lack of an adequate vascularization. Conversely, in Id1+/- Id3-/- mice transplanted with wild-type Id1+/+ Id3+/+ bone marrow cells, vasculogenesis and tumor progression were similar to wild-type mice. In addition to these studies, de Bont et al. (4) found that mobilized human CD34+ progenitor cells enhance tumor growth in a preclinical murine lymphoma model, Bolontrade et al. (5) demonstrated that CEP-induced vasculogenesis plays a role in a preclinical murine model of Ewing’s sarcoma, and Shirakawa et al. (6) provided evidence of postnatal vasculogenesis in breast-cancer-bearing mice. Along this line, we have demonstrated recently that CECs and their CEP subsets increase in some preclinical cancer models (7, 8) , as well as in cancer patients (9) .

It is well known that during the break periods between successive cycles of MTD chemotherapy, including CTX, there can be a marked mobilization of hematopoietic progenitors from the marrow into the peripheral blood circulation, as part of an adaptive response to the chemotherapy induced myelosuppression (10) . This raises several questions. First, would the levels of CEPs mobilized also show a similar decrease followed by a rapid recovery and increase shortly after MTD chemotherapy; second, might it be the case that such increase are not observed when the same drug is administered more frequently at lower doses, i.e., metronomically? If so, this might be suggestive of a mechanism by which low-dose metronomic chemotherapy regimens suppress angiogenesis without causing an increase in the severity of undesirable side effects normally associated with traditional cytotoxic chemotherapy regimens, such as myelosuppression (11, 12, 13) . It may also explain part of the basis of the robust repair process to damaged tumor endothelial cells that takes place during the long break periods between successive cycles of MTD chemotherapy (11) .

The purpose of this study was to investigate the effects of MTD versus metronomic regimens of the same chemotherapeutic drug (CTX) on CEP kinetics and viability using two preclinical models of human lymphoma xenografts. In particular, we were interested in evaluating whether one of the mechanisms of the angiogenic effects of metronomic CTX might be a sustained suppression of the mobilization and/or viability of bone-marrow-derived CEPs. We found strikingly different effects on MTD versus metronomic CTX on CEP mobilization and viability, which could have implications for the clinical use of metronomic chemotherapy regimens should similar findings apply in patients.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Animal Models.
NOD/SCID mice of 6–8 weeks of age received i.p. injections of 10 x 106 Namalwa or Granta 519 cells (ATCC, Manassas, VA) as we have described previously (7, 8 , 14) . Namalwa cells derived from an EBV+ Burkitt’s lymphoma, Granta 519 cells were derived from a mantle cell lymphoma in leukemic transformation. Animals were evaluated for tumor growth every other day, tumors were measured by calipers, and the formula [width2 x length x 0.52] was applied for approximating the volume of a spheroid (7, 8) . Tumor-bearing NOD/SCID mice (n = 12 per study group), were treated with CTX at the MTD for NOD/SCID mice (75 mg/kg on days 3, 5 and 7, or 225 mg/kg/cycle of therapy repeated every 21 days; see Ref. 12 ) or a metronomic low-dose schedule dose according to Browder et al. (170 mg/kg every 6 days; see Ref. 11 ) i.p. in a site remote from the inoculated tumor or a metronomic low-dose schedule of CTX in drinking water according to Man et al. (~20 mg/kg/day, Ref. 12 ). Circulating levels of VEGF were measured by a commercially available assay as reported previously (7) .

All procedures involving animals were performed in accordance with national and international laws and policies.

CEC and CEP Measurement by FC.
Mice were bled from retro-orbital sinus for CEC and CEP evaluation, which was performed by enumeration using four-color FC. Monoclonal antibodies reacting with CD45 were used to exclude hematopoietic cells; CECs and their CEP subset were depicted as described previously (7, 8) using the endothelial murine markers VEGF receptor 2 fetal liver kinase 1, CD13, and CD117 (PharMingen BD, San Diego, CA). Nuclear staining (Procount; BD Biosciences, San Jose, CA) was used on some occasions to exclude the possibility that platelets or cell debris hampered the accuracy of CEC and CEP enumeration (8, 9) . After RBC lysis, cell suspensions were evaluated by a FACSCalibur (BD Biosciences) using analysis gates designed to exclude dead cells, platelets, and debris. After acquisition of at least 100,000 cells/sample, analyses were considered as informative when adequate numbers of events (i.e., >50, typically 100–200) were collected in the CEC and CEP enumeration gates. Percentages of stained cells were determined and compared with appropriate negative controls. Positive staining was defined as being greater than nonspecific background staining, and 7AAD was used to enumerate viable, apoptotic, and dead cells (15) .

Evaluation of CEP Clonogenic Potential.
At the time of sacrifice, animals were evaluated for the presence of clonogenic CEP as we described previously (8) . A total of 0.5 x 106 nucleated cells were seeded in Petri dishes coated previously with fibronectin in the presence of collagen gel, EC medium, 12.5% FCS and 12.5% horse serum supplemented with VEGF (100 ng/ml) and basic fibroblast growth factor (5 ng/ml). Cells were cultured at 37°C. After 2 weeks of culture, colonies with endothelial morphology (elongated, sprouting or spindle cells) were enumerated and sub-clones established by picking colonies and resuspending the cells in the presence of VEGF and basic fibroblast growth factor. Fresh medium and cytokines were added weekly. After a 4-week culture, when seeded cells showed endothelial characteristics (patterned, tubular networks, sometimes multinucleate cells), the endothelial phenotype (CD45-, CD31+) of cultured cells was evaluated and confirmed by FC.

Statistical Analysis.
Statistical comparisons were performed using the t test, ANOVA, and linear regression when data were normally distributed, and the nonparametric analyses of Spearman and Mann-Whitney when data were not normally distributed. Values of P < 0.05 were considered statistically significant.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
CEC and CEP enumeration by FC are depicted in Fig. 1Citation . In our previous work (7, 8) , CECs were enumerated as CD45-VEGFR2+ cells, and the CEP subset was enumerated as CD45-VEGFR2+CD117+ cells. Consistently with what we have observed previously (8) , CD117 expression was found in 29–88% of CEC. In the present study, we found that the frequency of CD45-VEGFR2+CD117+ CEP was similar to that of CD45-CD13+ CD117+ CEP (Fig. 1, D and E)Citation . Furthermore, as shown in Fig. 1Citation , >98% of CD45-CD13+ CECs were CD45-VEGFR2+. In addition to the hematopoietic compartment, CD13 is also found in nonhematopoietic tissues, and its novel expression by endothelial cells of angiogenic, but not normal, vasculature has been described recently (16) . Treatment of animals with CD13 inhibitors significantly impaired neovascularization and xenograft tumor growth, indicating that CD13 plays an important functional role in vasculogenesis and identifying it as a critical regulator of angiogenesis (16) . VEGFR2+CD13+ cells included 40–100% of the CD45- cell population. In the CD45- cell fraction, the frequency of cells that expressed VEGFR2 but not CD13 (or vice versa) was always <5%. Regarding CD117 (c-kit), the crucial role of c-kit expression in hematopoietic and endothelial progenitors has been recently underlined by Heissig et al. (17) .



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  		Fig. 1. A–G, representative FC evaluation of CEC and CEP enumeration and viability. A shows the gate used to exclude platelets, dead cells, and debris. B shows the Ig-PE and Ig-PerCP negative controls. C shows the Ig-FITC and Ig-PerCP negative controls. D and E show that >98% of CD45-CD13+ (D) CECs were CD45- VEGFR2+ (E). F shows the gate on CD45-CD13+ cells used to evaluate CEC (CD45-CD13+CD117-) and CEP (CD45-CD13+CD117+) frequency and their viability according to 7AAD expression in G and H. Panels represent the same sample evaluated with negative controls (A–C), FITC-anti-CD13, PE-anti-VEGFR2, PerCP-anti-CD45, and APC-anti-CD117 (D and E), and FITC-anti-CD13, PE-anti-CD45, 7AAD, and APC-anti-CD117 (F–H).

 
To study CEP viability, considering that a FITC-conjugated anti-VEGFR2 antibody was not available for the present studies, we enumerated viable CEP as CD45-CD13+ CD117+7AAD- cells. The relative frequency of CEC and CEP in tumor-free NOD/SCID mice evaluated as controls and in tumor-bearing mice evaluated at the time of sacrifice is indicated in Fig. 2Citation . CEP frequency and viability was significantly increased in tumor-bearing animals (P < 0.01).



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  		Fig. 2. Top, frequency of CD117- (CEC) and CD117+ (CEP) CD45-CD13+ cells in tumor-free NOD/SCID mice evaluated as controls and in Namalwa tumor-bearing mice evaluated at sacrifice. Bottom, frequency of viable CEC and CEP in tumor-free and tumor-bearing NOD/SCID mice.

 
As shown in Figs. 3Citation (Namalwa-bearing mice) and 4Citation (Granta 519-bearing mice), CEP increase in untreated animals paralleled tumor growth in both models of human lymphoma. As described previously (7, 8, 9 , 18) , this might be attributable to the increase of tumor-derived VEGF production paralleling tumor growth in size. As we observed and reported in our previous study (7) , a positive correlation was found in tumor-bearing mice between CEC and human VEGF (r = 0.532, P = 0.04). However, it is unlikely that the increase in VEGF levels can itself explain CEP mobilization kinetics. In fact, marrow homing and mobilization in the peripheral blood of hematopoietic and/or endothelial progenitors is known to rely on complex (and still poorly understood) molecular pathways where integrins and multiple cell-to-cell and cell-to-matrix machinery, in addition to cytokine and chemokine levels, play a dominant role (19, 20) . We are currently measuring VEGF levels in cancer patients treated with MTD and metronomic CTX to better understand the relative contribution of this cytokine in CEP mobilization.



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  		Fig. 3. Tumor volume, day of MTD (gray arrows) and metronomic (black arrows) treatment and CEP kinetics in Namalwa-injected NOD/SCID mice. The panel on the right indicates the frequency of viable (7AAD-) CEC and CEP on day 20. Results are expressed as means ± 1 SD (bars); *P < 0.01.

 


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  		Fig. 4. Tumor volume, day of MTD (gray arrows) and metronomic (black arrows) treatment and CEP kinetics in Granta 519-injected NOD/SCID mice. The panel on the right indicates the frequency of viable (7AAD-) CEC and CEP on day 20. Results are expressed as means ± 1 SD (bars); *P < 0.01; **P < 0.001.

 
In both lymphoma models, the first course of MTD CTX delayed but did not prevent tumor growth, and a dramatic increase in CEP was observed a few days after MTD CTX. The CEP increase after MTD was not attributable solely to tumor growth, because, in both models, its magnitude was significantly higher than that observed in control animals, which, at the same time, already had significantly larger tumors. In MTD CTX-treated animals, the subsequent courses of MTD CTX did not reduce tumor growth. In marked contrast, in metronomic CTX-treated animals, CEPs were not increased, and their viability was significantly reduced when compared with MTD-treated animals (Figs. 3Citation and 4Citation ).

In comparison with MTD-treated animals, tumor growth was significantly delayed in Namalwa-bearing mice treated with metronomic CTX administered daily in drinking water. Tumor growth was not observed up to day 40 (i.e., 1 week after therapy discontinuation) in Namalwa-bearing mice treated with metronomic CTX administered on a weekly basis according the method of Browder et al. (170 mg/kg every 6 days; see Ref. 11 ). In these mice, when therapy was discontinued, CEP increase paralleled tumor growth, but the CEP mobilization effect observed after MTD CTX was not seen. In Granta 519-bearing mice, observed up to day 90, tumor growth (and, in turn, CEP increase) was abrogated by metronomic CTX [administered either according to Browder et al. (11) or according to Man et al. (12) ], but not by MTD CTX.

In CEP clonogenic assays, cultures of tumor-bearing mice treated with MTD CTX and evaluated at sacrifice generated a mean of 14 ± 7 colonies. In sharp contrast, cultures of tumor-bearing mice treated with metronomic CTX generated a mean of 1 ± 2 colonies (P = 0.001).


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
These results support the novel finding that MTD and metronomic chemotherapy have opposite effects on CEP mobilization and viability. MTD CTX mobilized viable CEPs, whereas metronomic CTX did not mobilize CEPs and, moreover, increased the frequency of apoptotic CEP. Although a formal demonstration of an obligatory role for CEP mediated vasculogenesis was not carried out in this study, it is highly likely that such cells do in fact make a significant contribution to the growth of the lymphoma models we have studied. For example, growth of other lymphoma cell lines is strongly suppressed in Id mutant mice, which are deficient in CEP-mediated vasculogenesis (3) , and previous studies in our laboratory have shown that continuous infusion of endostatin inhibits CEP mobilization and tumor growth in one preclinical lymphoma model (8) .

Our results strengthen the conclusion that the antitumor effects of low-dose metronomic chemotherapy are attributable, at least in part, to a mechanism involving inhibition of tumor blood vessel formation. In addition to antiangiogenic mechanisms in which fully differentiated endothelial cells are growth-inhibited and/or killed by metronomic low-dose chemotherapy (11) , an antivasculogenic process may also be involved that is mediated through effects on reducing CEP mobilization and viability. It is also interesting to consider whether MTD chemotherapy may sometimes accelerate tumor (re)growth and drug resistance by increased mobilization of CEPs. This may also help explain the robust reversal of the damage inflicted by MTD CTX on tumor blood vessel endothelial cells as noted by Browder et al. (11) . An influx of mobilized CEPs during the rest periods between cycles of MTD therapy may replace damaged or killed endothelial cells. In this regard, evaluating the mobilization, viability, and levels of CEPs detected in cancer patients treated with low-dose metronomic chemotherapy regimens, e.g., daily low-dose oral CTX and twice-a-week oral methotrexate in breast cancer (21) or leukeran in lymphoma4 will be of considerable interest. Such studies, which we are currently undertaking, may provide a surrogate marker to monitor the antivasculogenic effects of metronomic chemotherapy protocols.


   ACKNOWLEDGMENTS
 
We thank Aron Goldhirsch and Giovanni Martinelli for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by AIRC (Associazione Italiana per la Ricerca sul Cancro) and FIRC (Fondazione Italiana per la Ricerca sul Cancro). F. B. is a scholar of the United States National Blood Foundation. R. S. K. is a recipient of a Tier I Canada Research Chair and is supported by NIH Grant CA-41223. Back

2 To whom requests for reprints should be addressed, at Division of Hematology-Oncology European Institute of Oncology via Ripamonti 435, 20141 Milan, Italy. Phone: 39-02-57489535; Fax: 39-02-57489537; E-mail: francesco.bertolini{at}ieo.it Back

3 The abbreviations used are: CEP, circulating endothelial progenitor; CTX, cyclophosphamide; MTD, maximum tolerable dose; CEC, circulating endothelial cell; VEGF, vascular endothelial growth factor; FC, flow cytometry; 7AAD, 7-aminoactinomycin D. Back

4 P. Mancuso and F. Bertolini, manuscript in preparation. Back

Received 3/ 4/03. Accepted 6/13/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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Phase II study of metronomic chemotherapy for recurrent malignant gliomas in adults
Neuro-oncol, July 1, 2007; 9(3): 354 - 363.
[Abstract] [Full Text] [PDF]

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J. C. McAuliffe and J. C. Trent
Biomarkers in Gastrointestinal Stromal Tumor: Should We Equate Blood-Based Pharmacodynamics with Tumor Biology and Clinical Outcomes?
Clin. Cancer Res., May 1, 2007; 13(9): 2535 - 2536.
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C. Folkins, S. Man, P. Xu, Y. Shaked, D. J. Hicklin, and R. S. Kerbel
Anticancer Therapies Combining Antiangiogenic and Tumor Cell Cytotoxic Effects Reduce the Tumor Stem-Like Cell Fraction in Glioma Xenograft Tumors
Cancer Res., April 15, 2007; 67(8): 3560 - 3564.
[Abstract] [Full Text] [PDF]

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 haematolHome page
Y. Oki and A. Younes
Endothelial progenitor cells in non-Hodgkin's lymphoma
Haematologica, April 1, 2007; 92(4): 433 - 434.
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Q. Zhou, P. Guo, X. Wang, S. Nuthalapati, and J. M. Gallo
Preclinical Pharmacokinetic and Pharmacodynamic Evaluation of Metronomic and Conventional Temozolomide Dosing Regimens
J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 265 - 275.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
A. A. Kamat, T. J. Kim, C. N. Landen Jr., C. Lu, L. Y. Han, Y. G. Lin, W. M. Merritt, P. H. Thaker, D. M. Gershenson, F. Z. Bischoff, et al.
Metronomic Chemotherapy Enhances the Efficacy of Antivascular Therapy in Ovarian Cancer
Cancer Res., January 1, 2007; 67(1): 281 - 288.
[Abstract] [Full Text] [PDF]

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 The OncologistHome page
A. Suvannasankha, C. Fausel, B. E. Juliar, C. T. Yiannoutsos, W. B. Fisher, R. H. Ansari, L. L. Wood, G. G. Smith, L. D. Cripe, and R. Abonour
Final Report of Toxicity and Efficacy of a Phase II Study of Oral Cyclophosphamide, Thalidomide, and Prednisone for Patients with Relapsed or Refractory Multiple Myeloma: A Hoosier Oncology Group Trial, HEM01-21
Oncologist, January 1, 2007; 12(1): 99 - 106.
[Abstract] [Full Text] [PDF]

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 ScienceHome page
Y. Shaked, A. Ciarrocchi, M. Franco, C. R. Lee, S. Man, A. M. Cheung, D. J. Hicklin, D. Chaplin, F. S. Foster, R. Benezra, et al.
Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors.
Science, September 22, 2006; 313(5794): 1785 - 1787.
[Abstract] [Full Text] [PDF]

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R. Buckstein, R. S. Kerbel, Y. Shaked, R. Nayar, C. Foden, R. Turner, C. R. Lee, D. Taylor, L. Zhang, S. Man, et al.
High-Dose Celecoxib and Metronomic "Low-dose" Cyclophosphamide Is an Effective and Safe Therapy in Patients with Relapsed and Refractory Aggressive Histology Non-Hodgkin's Lymphoma
Clin. Cancer Res., September 1, 2006; 12(17): 5190 - 5198.
[Abstract] [Full Text] [PDF]

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N. Mehra, M. Penning, J. Maas, L. V. Beerepoot, N. van Daal, C. H. van Gils, R. H. Giles, and E. E. Voest
Progenitor Marker CD133 mRNA Is Elevated in Peripheral Blood of Cancer Patients with Bone Metastases.
Clin. Cancer Res., August 15, 2006; 12(16): 4859 - 4866.
[Abstract] [Full Text] [PDF]

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A. Bottini, D. Generali, M. P. Brizzi, S. B. Fox, A. Bersiga, S. Bonardi, G. Allevi, S. Aguggini, G. Bodini, M. Milani, et al.
Randomized Phase II Trial of Letrozole and Letrozole Plus Low-Dose Metronomic Oral Cyclophosphamide As Primary Systemic Treatment in Elderly Breast Cancer Patients
J. Clin. Oncol., August 1, 2006; 24(22): 3623 - 3628.
[Abstract] [Full Text] [PDF]

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S. S.W. Ng, A. Sparreboom, Y. Shaked, C. Lee, S. Man, N. Desai, P. Soon-Shiong, W. D. Figg, and R. S. Kerbel
Influence of Formulation Vehicle on Metronomic Taxane Chemotherapy: Albumin-Bound versus Cremophor EL-Based Paclitaxel.
Clin. Cancer Res., July 15, 2006; 12(14): 4331 - 4338.
[Abstract] [Full Text] [PDF]

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 BloodHome page
P. Mancuso, M. Colleoni, A. Calleri, L. Orlando, P. Maisonneuve, G. Pruneri, A. Agliano, A. Goldhirsch, Y. Shaked, R. S. Kerbel, et al.
Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy
Blood, July 15, 2006; 108(2): 452 - 459.
[Abstract] [Full Text] [PDF]

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R. S. Kerbel
Antiangiogenic therapy: a universal chemosensitization strategy for cancer?
Science, May 26, 2006; 312(5777): 1171 - 1175.
[Abstract] [Full Text] [PDF]

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S. D. Young, M. Whissell, J. C.S. Noble, P. O. Cano, P. G. Lopez, and C. J. Germond
Phase II Clinical Trial Results Involving Treatment with Low-Dose Daily Oral Cyclophosphamide, Weekly Vinblastine, and Rofecoxib in Patients with Advanced Solid Tumors.
Clin. Cancer Res., May 15, 2006; 12(10): 3092 - 3098.
[Abstract] [Full Text] [PDF]

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R. Munoz, S. Man, Y. Shaked, C. R. Lee, J. Wong, G. Francia, and R. S. Kerbel
Highly Efficacious Nontoxic Preclinical Treatment for Advanced Metastatic Breast Cancer Using Combination Oral UFT-Cyclophosphamide Metronomic Chemotherapy.
Cancer Res., April 1, 2006; 66(7): 3386 - 3391.
[Abstract] [Full Text] [PDF]

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M. Franco, S. Man, L. Chen, U. Emmenegger, Y. Shaked, A. M. Cheung, A. S. Brown, D. J. Hicklin, F. S. Foster, and R. S. Kerbel
Targeted Anti-Vascular Endothelial Growth Factor Receptor-2 Therapy Leads to Short-term and Long-term Impairment of Vascular Function and Increase in Tumor Hypoxia.
Cancer Res., April 1, 2006; 66(7): 3639 - 3648.
[Abstract] [Full Text] [PDF]

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Y. Shaked, U. Emmenegger, S. Man, D. Cervi, F. Bertolini, Y. Ben-David, and R. S. Kerbel
Optimal biologic dose of metronomic chemotherapy regimens is associated with maximum antiangiogenic activity
Blood, November 1, 2005; 106(9): 3058 - 3061.
[Abstract] [Full Text] [PDF]

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R. Yap, D. Veliceasa, U. Emmenegger, R. S. Kerbel, L. M. McKay, J. Henkin, and O. V. Volpert
Metronomic Low-Dose Chemotherapy Boosts CD95-Dependent Antiangiogenic Effect of the Thrombospondin Peptide ABT-510: A Complementation Antiangiogenic Strategy
Clin. Cancer Res., September 15, 2005; 11(18): 6678 - 6685.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
Y. Shaked, U. Emmenegger, G. Francia, L. Chen, C. R. Lee, S. Man, A. Paraghamian, Y. Ben-David, and R. S. Kerbel
Low-dose Metronomic Combined with Intermittent Bolus-dose Cyclophosphamide Is an Effective Long-term Chemotherapy Treatment Strategy
Cancer Res., August 15, 2005; 65(16): 7045 - 7051.
[Abstract] [Full Text] [PDF]

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 Ann OncolHome page
G. Bocci, M. Tuccori, U. Emmenegger, V. Liguori, A. Falcone, R. S. Kerbel, and M. Del Tacca
Cyclophosphamide-methotrexate 'metronomic' chemotherapy for the palliative treatment of metastatic breast cancer. A comparative pharmacoeconomic evaluation
Ann. Onc., August 1, 2005; 16(8): 1243 - 1252.
[Abstract] [Full Text] [PDF]

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M. Loeffler, J. A. Kruger, and R. A. Reisfeld
Immunostimulatory Effects of Low-Dose Cyclophosphamide Are Controlled by Inducible Nitric Oxide Synthase
Cancer Res., June 15, 2005; 65(12): 5027 - 5030.
[Abstract] [Full Text] [PDF]

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Y. Shaked, D. Cervi, M. Neuman, L. Chen, G. Klement, C. R. Michaud, M. Haeri, B. J. Pak, R. S. Kerbel, and Y. Ben-David
The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells
Blood, June 1, 2005; 105(11): 4500 - 4507.
[Abstract] [Full Text] [PDF]