Sergey V. Kozin

Vascular Normalization by Vascular Endothelial Growth Factor Receptor 2 Blockade Induces a Pressure Gradient Across the Vasculature and Improves Drug Penetration in Tumors

Elevated interstitial fluid pressure, a hallmark of solid tumors, can compromise the delivery of therapeutics to tumors. Here we show that blocking vascular endothelial growth factor (VEGF) signaling by DC101 (a VEGF-receptor-2 antibody) decreases interstitial fluid pressure, not by restoring lymphatic function, but by producing a morphologically and functionally “normalized” vascular network. We demonstrate that the normalization process prunes immature vessels and improves the integrity and function of the remaining vasculature by enhancing the perivascular cell and basement membrane coverage. We also show that DC101 induces a hydrostatic pressure gradient across the vascular wall, which leads to a deeper penetration of molecules into tumors. Thus, vascular normalization may contribute to the improved survival rates in tumor-bearing animals and in colorectal carcinoma patients treated with an anti-VEGF antibody in combination with cytotoxic therapies.

The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis

Gliomas are the most common primary tumours of the central nervous system, with nearly 15,000 diagnosed annually in the United States and a lethality approaching 80% within the first year of glioblastoma diagnosis. The marked induction of angiogenesis in glioblastomas suggests that it is a necessary part of malignant progression; however, the precise molecular mechanisms underlying the regulation of brain tumour growth and angiogenesis remain unresolved. Here we report that a candidate tumour suppressor gene, ING4, is involved in regulating brain tumour growth and angiogenesis. Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. In mice, xenografts of human glioblastoma U87MG, which has decreased expression of ING4, grow significantly faster and have higher vascular volume fractions than control tumours. We show that ING4 physically interacts with p65 (RelA) subunit of nuclear factor NF-κB, and that ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-κB-responsive genes. These results indicate that ING4 has an important role in brain tumour pathogenesis.

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.

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels

Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-β1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors —inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer therapeutics.

Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics

The extracellular pH of tumor tissue is significantly lower than the extracellular pH of normal tissue, whereas the intracellular pH of both tissues is similar. In principle, extracellular acidity may be expected to enhance the intracellular uptake and cytotoxicity of weak acid chemotherapeutics that are membrane permeable in their uncharged state and inhibit the efficacy of weak bases. However, procedures for assessing the role of the gradient as a determinant of drug efficacy in vivo by altering the pH gradient may also alter drug availability and thus mask or exaggerate the effect of the gradient change. In the present study, we have altered the extracellular pH of tumors and compared the effect of the resultant pH gradient change on the efficacy of a weak acid versus a weak base. This experimental design gives rise to a change in the ratio of chlorambucil- to doxorubicin-induced tumor growth delay, independent of possible changes in drug availability. The extracellular pH of the 54A human tumor in NCr/Sed/nu/nu mice was altered by administration of 5 mg/g i.v. glucose. The resultant 0.2 pH unit increase in the tumor cell pH gradient gives rise to a predicted 2.3-fold increase in the ratio of chlorambucil to doxorubicin growth delay. The experimentally measured change in the growth delay ratio was 2.1. The results provide compelling evidence that the pH gradient in a determinant of the efficacy of weak electrolytes in the complex in vivo environment and may be exploited for the treatment of cancer. [Mol Cancer Ther 2006;5(5):1275–9]

Relative biological effectiveness of proton beams in clinical therapy

PURPOSE In clinical proton beam radiation therapy, an RBE of 1.1 relative to megavoltage X-rays is currently being employed at most treatment centers. This RBE pertains to radiation in the spread out Bragg-peak (SOBP) for all tissue systems, all dose levels per fraction and all proton beam energies. As the number of centers and treatment sites for which proton beam therapy continues to increase and additional experimental data is accrued, a re-assessment of the justification for a generic RBE is warranted. In this paper we address: (1) the constancy of the RBE along the central axis from the plateau entrance to the distal SOBP (upstream of the distal edge); (2) RBE as a function of dose (or cell survival level); and (3) the target cell or tissue (alpha/beta) dependency of the RBE. This analysis pertains to modulated proton beams of initial energies of approximately 70-200 MeV and SOBPs of approximately 2-10 cm, respectively. RESULTS AND CONCLUSIONS With exceptions, the available experimental data indicate that the RBE of SOBP protons increases with decreasing dose or dose per fraction and increasing depth in the SOBP, with the magnitude of both effects likely being dependent on the alpha/beta ratios of the target cells or tissues. The use of a generic RBE of 1. for all tissues, especially those exhibiting low alpha/beta values such as CNS, may be too low, especially at dose levels of < or = 2 Gy/fraction. Systematic determination of the RBE values dependent upon the three interdependent variables identified in this manuscript (beam depth, dose size and target tissue) will provide an enhanced data base for detailed treatment planning and institutional trial comparisons, thereby maximizing the therapeutic benefit of proton beams.

Perivascular nitric oxide gradients normalize tumor vasculature

Normalization of tumor vasculature is an emerging strategy to improve cytotoxic therapies. Here we show that eliminating nitric oxide (NO) production from tumor cells via neuronal NO synthase silencing or inhibition establishes perivascular gradients of NO in human glioma xenografts in mice and normalizes the tumor vasculature, resulting in improved tumor oxygenation and response to radiation treatment. Creation of perivascular NO gradients may be an effective strategy for normalizing abnormal vasculature.

The pH partition theory predicts the accumulation and toxicity of doxorubicin in normal and low-pH-adapted cells.

SummaryThe accumulation and toxicity of the weak base doxorubicin has been investigated as a function of extracellular pH, intracellular pH and the cellular pH gradient in cells previously cultured under normal (pH 7.4) and low-pH (6.8) conditions. Low-pH-adapted cells exhibit transmembrane pH gradients which substantially differ from normal cells at the same extracellular pH. No relationship was obtained between intracellular pH and the uptake or toxicity of doxorubicin in the two cell types. In contrast, doxorubicin accumulation and toxicity increased with increasing extracellular pH in both normal and low-pH-adapted cells. However, at the same extracellular pH, drug cytotoxicity was more pronounced in normal than in low-pH-adapted cells. The difference in doxorubicin accumulation and cytotoxicity at the same extracellular pH was found to be dependent on the difference in the transmembrane pH gradient of the two cell types. As the cellular pH gradient differs between tumour and normal tissue, this observation suggests a basis for enhancing cellular drug uptake in either tissue type.

Neovascularization After Irradiation: What is the Source of Newly Formed Vessels in Recurring Tumors?

Local relapse of tumors after radiation therapy remains a challenge in oncology. To devise rational approaches for preventing this relapse, we have to improve our understanding of how new vessels form in previously irradiated tumors. We propose that tumor regrowth after local irradiation is dependent on blood vessel formation by local endothelial cells without the need for recruitment of endothelial precursor cells from distant nonirradiated tissues or bone marrow. We also suggest that infiltrating myeloid bone marrow-derived cells promote survival of local endothelial cells during the early period after irradiation and angiogenesis during the later stage of tumor regrowth, both via paracrine mechanisms.

Blockade of MMP14 Activity in Murine Breast Carcinomas: Implications for Macrophages, Vessels, and Radiotherapy

BACKGROUND Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.