Lisa Volk

Lymphangiogenesis and Lymphatic Metastasis in Breast Cancer

Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.

Anti-VEGF-A therapy reduces lymphatic vessel density and expression of VEGFR-3 in an orthotopic breast tumor model.

Because metastasis contributes significantly to cancer mortality, understanding its mechanisms is crucial to developing effective therapy. Metastasis is facilitated by lymphangiogenesis, the growth of new intratumoral or peritumoral lymphatic vessels from pre‐existing vessels. Vascular endothelial growth factor A (VEGF‐A) is a well‐known angiogenic factor. Increasing evidence implicates VEGF‐A in lymphangiogenesis, although the mechanism of its pro‐lymphangiogenic effect is poorly understood. We examined the effect of the anti‐VEGF‐A neutralizing antibody 2C3 on tumor lymphangiogenesis and metastasis in an orthotopic breast carcinoma model using MDA‐MB‐231 cells and its luciferase‐tagged derivative, 231‐Luc+ cells. Anti‐VEGF‐A antibody therapy reduced blood and lymphatic vessel densities by 70% and 80%, respectively, compared with the control antibody. Treatment with 2C3 antibody also decreased incidence of lymphatic and pulmonary metastases by 3.2‐ and 4.5‐fold, respectively. Macrophage infiltration was reduced in 2C3‐treated tumors by 32%, but VEGF‐C expression was unchanged. In contrast, neoplastic cells and blood vessels in tumors from 2C3‐treated mice expressed significantly less angiopoietin‐2 (Ang‐2) than tumors from control mice. The reduction in Ang‐2 was associated with inhibition of VEGFR‐3 expression in intratumoral lymphatic endothelial cells. Both VEGF‐A and Ang‐2 upregulated the expression of VEGFR‐3 in cultured lymphatic endothelial cells. VEGF‐A induced proliferation of lymphatic endothelial cells was reduced by 50% by soluble Tie‐2, suggesting that Ang‐2 is an intermediary of the pro‐lymphangiogenic VEGF‐A effect. These results suggest a novel mechanism by which anti‐VEGF‐A therapy may suppress tumor lymphangiogenesis and subsequent metastasis supporting the use of anti‐VEGF‐A therapy to control metastasis clinically.

Tumor suppressor function of Rab25 in triple‐negative breast cancer

Rab proteins are a group of ubiquitously expressed proteins that are responsible for intracellular transport of vesicles. Recent evidence has shown that certain Rab proteins are involved in the pathogenesis of cancer. We have recently shown that Rab25 is lost in a large fraction of breast cancer samples, particularly those derived from hormonally insensitive tumors. We have further investigated the role of Rab25 by re‐expressing Rab25 in tumorigenic cell lines and measuring the impact on tumor formation as well as on various molecular pathways through PCR array analysis. In vivo tumor growth of cell lines with re‐expressed Rab25 was markedly suppressed. Our data suggest that Rab25 acts through multiple pathways to enhance apoptosis and to suppress angiogenesis and invasion by modulating VEGF‐A and VEGFR‐1 expression. These findings suggest that Rab25 represents a novel class of cellular modulators that can influence both tumor initiation and the progression of the established tumors, thus ultimately affecting the biology of the malignant disease.

Characterization of Prox1 and VEGFR-3 Expression and Lymphatic Phenotype in Normal Organs of Mice Lacking p50 Subunit of NF-κB

Please cite this paper as: Flister, Volk and Ran (2011). Characterization of Prox1 and VEGFR‐3 Expression and Lymphatic Phenotype in Normal Organs of Mice Lacking p50 Subunit of NF‐κB. Microcirculation18(2), 85–101.

Antitumor activity, and antiangiogenic activity of nanoparticle albumin-bound nab-rapamycin in combination with nab-paclitaxel.

Abstract #3125 Background: Rapamycin inhibits downstream signals from the mammalian target of rapamycin (mTOR), a known kinase member of a signaling pathway that promotes tumor growth. Rapamycin9s poor aqueous solubility and poor chemical stability have limited its development as an intravenous (IV) anticancer agent. Nab -rapamycin utilizes the albumin-bound technology to allow for IV administration of rapamycin and has demonstrated dose-linear pharmacokinetics and safety up to 90 mg/kg with effective antitumor activity at 40 mg/kg against a human panel of tumor xenografts. This study investigated the efficacy of combined therapy with nab -paclitaxel (Abraxane®) utilizing invasive human breast (MDA-MB-231) and colon (HT29) cancer xenograft models.
 Material and Methods: Xenograft transplants using luciferase-tagged MDA-MB-231 cells were implanted into mammary fatpad of SCID mice and allowed to reach 460 mm 3 in size prior to IV administration of saline (vehicle, n = 9); nab -rapamycin, 3x wkly for 2 wks at 40 mg/kg ( nab -rap-2W; n = 8); nab -rapamycin, 3x wkly for 4 wks at 40 mg/kg ( nab -rap-4W; n = 8); Abraxane, qdx5 at 30 mg/kg (ABX; n = 8); nab -rap-2W + ABX (n = 9); or nab -rap-4W + ABX (n = 8). HT29 xenografts were also treated with nab -rap-4W (n = 8) and nab -rap-4W + ABX (10 mg/kg, qdx5, IP, n = 8). The in vivo antiangiogenic effect of nab -rapamycin was evaluated using the standard in vivo chick chorioallantoic membrane (CAM) assay with 3-day old embryos (n = 18).
 Results: Relative to vehicle controls, nab -rap-2W ( P nab -rap-4W ( P P nab -rapamycin + ABX with TGI of 81% and 86% for nab -rap-2W + ABX and nab -rap-4W + ABX groups, respectively. For HT29 tumors, the combination of ABX and nab -rapamycin also showed greater TGI (89%) compared to nab -rapamycin alone (81%). In the chick CAM assay, nab -rapamycin demonstrated antiangiogenic efficacy at doses of 10 µg and above without affecting embryo viability.
 Conclusions: Combination therapy of nab -rapamycin and Abraxane was more effective at inhibiting breast and colon xenograft tumor growth than single therapy of either drug. The enhanced antitumor activity seen with combined nab -rapamycin-Abraxane may in part be due to the observed antiangiogenic activity of nab -rapamycin. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3125.

Abstract 3852: Combination treatment of nab-paclitaxel and bevacizumab in a new model of triple-negative breast cancer

Background: Tumors that lack estrogen, progesterone, and Her2/nu (triple-negative) are one of the most aggressive, therapy-resistant and highly metastatic subtypes of breast cancer (BC). Most currently available models do not recapitulate expression profile of triple-negative BC, thus making it difficult to devise new treatments that target this tumor type. Here, we established and characterized a new model of triple-negative BC. We also tested the novel combination of nab-paclitaxel with bevacizumab, which has been successful in treating other metastatic cancer types. Methods: HCC1806 cells were obtained stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase). The new derivative was designated as HCC1806-RR and characterized in proliferation and cytotoxicity assays. HCC1806-RR was also orthotopically implanted into mammary fat pads of immunodeficient mice to determine tumor cell sensitivity to chemotherapy alone or in combination with anti-VEGF-A antibody. Mice bearing orthotopic HCC1806-RR tumors of 150mm 3 in size were treated with saline (control), bevacizumab (4mg/kg. i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or with combination of nab-paclitaxel and bevacizumab. Tumor growth rate was monitored by using calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: The parental HCC1806 and its derivative HCC1806-RR had identical morphology, proliferation rates and sensitivity to nab-paclitaxel. Luciferase measurements and imaging in vivo showed that HCC1806-RR tumors have predominant LN metastasis with lungs being a second metastatic site. Bevacizumab and nab-paclitaxel inhibited tumor growth by 0% and 90%, respectively. Combination therapy inhibited tumor growth by 100%. This result was highly significant compared with either control (P Conclusions: The HCC1806-RR is a new model double-tagged with RFP and Renilla luciferase. These tags allow for quantitative assessment of metastatic spread. This model can be used to study the biology of triple-negative breast cancer and for designing new approaches to treat this type of cancer. Preliminary studies demonstrated high sensitivity of HCC1806-RR to nab-paclitaxel combined with bevacizumab suggesting that this therapy could significantly improve the health outcome for patients with triple-negative breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3852.

Abstract P1-03-11: Combination of nab-Paclitaxel and Bevacizumab Inhibited Tumor Growth and Metastasis of a New Triple Negative Breast Cancer Model

Background: Triple-negative breast cancers are highly aggressive and resistant to chemotherapy. In this study, we established a new model of triple-negative breast cancer and used it to test the novel combination of nab-paclitaxel with anti-VEGF antibody bevacizumab, which has been effective in treating other metastatic cancer types. Methods: The triple-negative HCC1806 beast cancer cells were stably transfected with dual reporters Red Fluorescent Protein (RFP) and Renilla luciferase to establish the new HCC1806-RR model. Immunodeficient mice bearing orthotopic HCC1806-RR tumors of 150mm 3 in size were treated with saline (control), bevacizumab (4 mg/kg. i.p., twice a week, for 10 weeks), nab-paclitaxel (10 mg/kg, i.v., qdx5), or with combination of nab -paclitaxel and bevacizumab. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: The HCC1806-RR cells had identical morphology, proliferation rates and sensitivity to nab-paclitaxel as the parental HCC1806 cell line. HCC1806-RR tumors primarily metastasized to LN, with lung being a secondary metastatic site. Combination therapy of nab -paclitaxel and bevacizumab inhibited tumor growth by 100%, as compared to 0% for bevacizumab (P (P = 0.024). Importantly, only combination therapy reduced incidence of LN and lung metastases by 50% (P = 0.007) and 87% (P = 0.001). Overall, 50% of the mice in the combination therapy group (n = 10) had complete regressions of both primary tumors and metastases at both regional and distant sites. Conclusions : The HCC1806-RR is a new triple-negative breast model with dual reporters to allow for quantitative assessment of metastatic spread. This model can be used to study the biology and treatment of triple-negative breast cancer. Combination of nab-paclitaxel with bevacizumab was highly effective against HCC 1806-RR tumors, suggesting that this regimen could provide clinical benefits to patients with triple-negative breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-11.

Abstract 3279: Nab-paclitaxel and bevacizumab treatment in new models of inflammatory breast cancer, SUM149-RR and SUM149-GL

Background: Inflammatory breast cancer (IBC) is a one of the most lethal forms of breast cancer with a 5-year survival of 40%. The hallmarks of IBC include skin redness, irritation, swelling, pain, as well as extensive lymph nodes (LN) and hematogenous metastasis. Animal models are highly desirable for studying the IBC pathology and for designing therapies that increase patient survival. Here we established and characterized two luciferase- and fluorescently-labeled lines derived from the human IBC line, SUM149. We characterized the growth and metastasis of these lines in vivo as well as their sensitivity to the novel drug combination, nab-paclitaxel and bevacizumab. Methods: SUM149 cells were stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase), or infected with lentivirus encoding Green Fluorescent Protein (GFP) and Firefly luciferase termed GL (GFP and Firefly Luciferase). The new cell lines (SUM149-RR and SUM149-GL) were characterized in vitro and in vivo after orthotopic implantation into mammary fat pads of immunodeficient mice. SUM149-RR was also examined for sensitivity to nab-paclitaxel alone or in combination with bevacizumab. Mice bearing tumors of 150mm3 in size were treated with saline (control), bevacizumab (4mg/kg i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or the combination. Tumor growth was monitored by calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: Luciferase measurements and in vivo imaging showed that both SUM149-RR and -GL clones were highly metastatic to LN, lungs, liver, brain, and spleen. Bevacizumab alone decreased tumor progression at later but not early stages of tumor growth. Nab-paclitaxel alone inhibited tumor growth by 73%. Combination therapy increased inhibition to 96%, and resulted in 22% (2/9) complete responses. SUM149-RR tumors in control mice displayed ulcerations, edema and redness much like the clinical disease. Tumors in mice treated with bevacizumab or combination therapy showed no signs of inflammation. Tumors from bevacizumab treated groups were more morphologically intact with reduced vascular abnormalities than tumors from control or nab-paclitaxel treated mice. LN and lung metastasis was significantly reduced in all treated groups as compared with control. Conclusions: The SUM149-RR and SUM149-GL lines are new double-tagged models for human IBC that allow organ visualization and accurate quantification of metastasis. These models can be used for better understanding of the IBC biology and for developing new therapies tailored to specific pathology of this cancer. Preliminary studies demonstrated high sensitivity of SUM149-RR to nab-paclitaxel/bevacizumab therapy suggesting the potential usefulness for treatment of IBC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3279.

Abstract 2354: Angiopoietin-2 role in breast cancer metastasis

Background: Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. The pro-angiogenic effect of Ang-2 is mediated through activation of the Tie-2 receptor that leads to vessel destabilization and dismantling. The latter is an essential step for both angiogenesis and tumor entry into the lumen. We assessed the effects of Ang-2 on tumor vasculature in orthotopic breast carcinoma model MDA-MB-231 that either lacked or overexpressed this factor. Methods: Luciferase-tagged MDA-MB-231 cells were engineered to overexpress human Ang-2. The control line expressed Ang-2 in reverse orientation. Stable production of Ang-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Ang-2 functionality was assessed by migration and proliferation assays using tumor and endothelial cells. Ang2-overexpressing and control MDA-MB-231 cells were orthotopically implanted into mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry using antibodies against MECA-32, smooth muscle actin (SMA-α), VEGFR-3 and Notch-1. Results: Ang-2-overexpressing tumor line produced 11±0.4 ng/ml of Ang-2 while expression in control line was undetectable. Tumor-produced Ang2 was functional as demonstrated by a dose-dependent migration of lymphatic endothelial cells. Soluble Tie-2 inhibited Ang-2 induced migration by 70%. Ang-2 had not effect on proliferation of MDA-MB-231 cells, tumor growth or blood vessel density. However, Ang-2 overexpressing and control tumors substantially differed in vascular morphology. The differences included 170% increase in blood vascular area, 280% increase in number of vessels with open lumen, and 600% increase in lumens’ cross-sectional area. Pericyte coverage shown by SMA-α staining decreased >10-fold (p>0.001) in Ang-2 tumors, demonstrating vessel destabilization. Tumor vascular invasion and pulmonary metastasis increased by 500% and 1100% in Ang-2 tumors compared with controls. Also, blood vessels in Ang-2 tumors displayed 2.0-2.2 fold upregulated Notch-1 and VEGFR-3 expression. Conclusions: These data suggest that Ang-2 increases metastasis via suppression of pericytes’ recruitment, which destabilizes tumor vessels and facilitates entry of tumor cells into the lumen. The increased expression of Notch-1 and VEGFR-3 on blood vessels in Ang-2 overexpressing tumors suggests that signaling of these proteins underlie the vascular morphologic changes in Ang-2-rich environment. This is consistent with prior data demonstrating up-regulation of VEGFR-3 on tumor blood vessels and association of both proteins with increased metastasis. This study demonstrates a key role for Ang-2 in hematogenous metastasis and suggests that suppression of Ang-2 and VEGFR-3 could inhibit breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2354.