Newer cancer therapies Angiotherapy use of agents that inhibit angiogenesis

Angiogenic therapy Rationale 1) tumour growth is angiogenesis-dependent 2) targets the genetically-stable microvascular endothelial cell 3) anti-angiogenic compounds are cytostatic

Composition of nascent and mature blood vessel walls Nascent vessels consist of a tube of ECs, which mature into specialized capillaries, arteries and veins. (b) Capillaries consist of ECs surrounded by basement membrane and a sparse layer of pericytes embedded within the EC basement membrane. Capillary endothelial layer can be continuous (muscle), fenestrated (kidney/ endocrine glands) or discontinuous (liver sinusoids). The endothelia of the blood-brain barrier or blood-retina barrier are further specialized to include tight junctions, and are thus impermeable to various molecules. (c) Arterioles and venules have an increased coverage of mural cells compared with capillaries. a) Nascent vessels consist of a tube of ECs. These mature into the specialized structures of capillaries, arteries and veins. (b) Capillaries, the most abundant vessels in our body, consist of ECs surrounded by basement membrane and a sparse layer of pericytes embedded within the EC basement membrane. Because of their wall structure and large surface-area-to-volume ratio, these vessels form the main site of exchange of nutrients between blood and tissue. Depending upon the organ or tissue, the capillary endothelial layer is continuous (as in muscle), fenestrated (as in kidney or endocrine glands) or discontinuous (as in liver sinusoids). The endothelia of the blood-brain barrier or blood-retina barrier are further specialized to include tight junctions, and are thus impermeable to various molecules. (c) Arterioles and venules have an increased coverage of mural cells compared with capillaries. Precapillary arterioles are completely invested with vascular SMCs that form their own basement membrane and are circumferentially arranged, closely packed and tightly associated with the endothelium. Extravasation of macromolecules and cells from the blood stream typically occurs from postcapillary venules

Steps in network formation and maturation during embryonic (physiological) angiogenesis Figure 2. Steps in network formation and maturation during embryonic (physiological) angiogenesis (a) and tumor (pathological) angiogenesis (b). (a) The nascent vascular network forms from an initial cell plexus by processes of vasculogenesis or angiogenesis. This is regulated by cell-cell and cell-matrix signaling molecules (Box 2) and mechanical forces, as is further growth and expansion of the network (by proliferating and migrating cells) alongside its normal remodeling by cell death (apoptosis). Ordered patterns of growth, organization and specialization (including the investment of vascular channels by mural cells) produce mature networks of arteries, capillaries and veins—networks that are structurally and functionally stable and appropriate to organ and location. (b) ECs and mural cells derived from circulating precursor cells or from the host vasculature form networks that are structurally and functionally abnormal. Continual remodeling by inappropriate patterns of growth and regression (cell apoptosis and necrosis) contribute to the instability of these networks.

Key differences in tumour vasculature Different flow characteristics / blood volume Microvasculature permeability Increased fractional volume of extravascular, extracellular space

Steps in network formation and maturation during tumour angiogenesis Figure 2. Steps in network formation and maturation during embryonic (physiological) angiogenesis (a) and tumor (pathological) angiogenesis (b). (b) ECs and mural cells derived from circulating precursor cells or from the host vasculature form networks that are structurally and functionally abnormal. Continual remodeling by inappropriate patterns of growth and regression (cell apoptosis and necrosis) contribute to the instability of these networks.

Cellular mechanisms of tumour angiogenesis (1) host vascular network expands by budding of endothelial sprouts or formation of bridges (angiogenesis); (2) tumour vessels remodel and expand by the insertion of interstitial tissue columns into the lumen of pre-existing vessels (intussusception); and (3) endothelial cell precursors (angioblasts) home from the bone marrow or peripheral blood into tumours and contribute to the endothelial lining of tumour vessels (vasculogenesis) (4) Lymphatic vessels around tumours drain the interstitial fluid and provide a gateway for metastasizing tumour cells. 3 2 1 4

Angiogenesis-overview Tumour cells release pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and bind to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. Such interactions between endothelial cells and tumour cells lead to the secretion and activation of various proteolytic enzymes, such as matrix metalloproteinases (MMPs), which degrade the basement membrane and the extracellular matrix. Degradation allows activated endothelial cells — which are stimulated to proliferate by growth factors — to migrate towards the tumour. Integrin molecules, such as v 3-integrin, help to pull the sprouting new blood vessel forward. The endothelial cells deposit a new basement membrane and secrete growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. PDGFR, PDGF receptor; VEGFR, VEGF receptor. Nature Reviews Drug Discovery 1, 415-426 (2002)

Angiogenesis-overview Balance between inhibitory factors (endostatin) and angiogenic factors (VEGF, bFGF) Tumour cells release pro-angiogenic factors which activate receptors (VEGFR) also stimulates secretion and activation of MMPs which degrade the basement membrane This allows activated endothelial cells to migrate towards tumour, helped by integrins ECs deposit a new basement membrane and secrete growth factors such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. Tumour cells release pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and bind to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. Such interactions between endothelial cells and tumour cells lead to the secretion and activation of various proteolytic enzymes, such as matrix metalloproteinases (MMPs), which degrade the basement membrane and the extracellular matrix. Degradation allows activated endothelial cells — which are stimulated to proliferate by growth factors — to migrate towards the tumour. Integrin molecules, such as v 3-integrin, help to pull the sprouting new blood vessel forward. The endothelial cells deposit a new basement membrane and secrete growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. PDGFR, PDGF receptor; VEGFR, VEGF receptor. VEGF – Vascular Endothelial Growth Factor bFGF - basic Fibroblast Growth Factor MMPs – Matrix MetalloProteinases

‘cryptic’ angiogenesis inhibitors Inactive until they are released from the parent protein by enzymatic cleavage Angiostatin 38kDa fragment of plasminogen Endostatin 20kDa fragment of collagen XVIII Endothelial cell specific Complete regression in mice No drug resistance

Endostatin Discovered in 1995 by Judah Folkman et al Phase I clinical trial in 1999 Dr. James Watson predicted that Dr. Folkman would cure all cancer within 2 years Dr. Folkman’s response “If you are a mouse and have cancer we can take good care of you. I respectfully disagree because in our experiments we mostly sacrifice the mice. So, I don't know if that qualifies as taking good care” www.pbs.org/wgbh/ nova/cancer/program.html

Integrins – the ‘velcro’ of the cell The cell moves by "ruffling" it's membrane. This is done by a series of actin fibers, whose function is controlled by the integrins. These fibers cause the cell membrane to move in certain directions, and the integrins attach to the matrix as this happens, pulling the cell along a micrometer at a time

Representation of the clinical drug development process suggested differences in end points between studies that are targeted at cytotoxic agents compared with studies to test angiogenic modulators DLT, dose-limiting toxicity; MTD, maximum-tolerated dose.

What is in the pipeline? Anti-angiogenic molecules fall into 5 categories inhibitors of pro-angiogenic growth factors, e.g. VEGF, bFGF, PDGF protease inhibitors that prevent the breakdown of the surrounding matrix, which is needed for blood-vessel growth; Analogs of endogenous inhibitors of angiogenesis e.g. endostatin; inhibitors of cellular adhesion molecules; and molecules with undefined mechanisms

Anti-VEGFR2 therapy (c,d) Anti-VEGFR2 prunes immature vessels, leading to a progressively 'normalized' vasculature (e) Further treatment leads to a vasculature that is inadequate to sustain tumour growth by day 5. (f) Perivascular cells expressing GFP (under the control of the VEGF promoter) envelope some vessels in the tumour interior. (g) A perivascular cell, presumably a fibroblast, leading the endothelial sprout (arrow). Vessel normalization and EC−mural cell interactions in tumors growing in dorsal windows in mice. (a) Normal capillary bed (dorsal skin and striated muscle). (b) Tumor vasculature (human tumor xenograft). (c,d) Anti-VEGFR2 therapy prunes immature vessels, leading to a progressively 'normalized' vasculature by days 1 and 2. (e) Further treatment leads to a vasculature that is inadequate to sustain tumor growth by day 5. (f) Perivascular cells expressing GFP (under the control of the VEGF promoter) envelope some vessels in the tumor interior. (g) A perivascular cell, presumably a fibroblast, leading the endothelial sprout (arrow). From ref. 53, with permission from Nature Medicine. Red, vessel lumens; green, perivascular cells. Scale bar (f,g) = 50 m. Images were obtained using a two-photon microscope.

Imaging studies to monitor tumour angiogenesis (blood flow) Before treatment after treatment Figure 3 | Imaging studies to monitor tumour angiogenesis. Blood-flow maps a | before treatment and b | six months after treatment of a patient with metastatic renal-cell carcinoma with thalidomide. The blood flow decreased from 127 ml min–1100 g–1 to 14 ml min–1100 g–1 of tissue. The arrows in the figure identify the area of the tumour. The colour coding indicates the range of blood flow: red–yellow, high blood flow; blue, low blood flow. Blood-flow maps a | before treatment and b | six months after treatment of a patient with metastatic renal-cell carcinoma with thalidomide.

Haematological malignancies! Although solid tumours are known to be angiogenesis dependent1, 114, it was assumed until 1993 (Ref. 143) that leukaemias and other haematological malignancies did not induce angiogenesis to promote their own survival. In 1993, Brunner et al. observed that basic fibroblast growth factor (bFGF) was expressed by human bone-marrow and peripheral-blood cells144, and the following year Nguyen et al. reported that bFGF was elevated in the urine of newly diagnosed leukaemic patients to higher levels than in most other malignancies145. Bone-marrow angiogenesis was subsequently found to correlate with multiple myeloma progression146-148, and was also observed in the lymph nodes of patients with B-cell non-Hodgkin's lymphoma149 and in bone-marrow biopsies from children with newly diagnosed untreated acute lymphoblastic leukaemia114. By 1999, cellular levels of another angiogenic protein — vascular endothelial growth factor — were reported to predict the outcome of patients with multiple myeloma150. The figure shows a comparison of normal versus leukaemic bone marrow, with blood vessels shown in red. These images are confocal microscopic sections of bone-marrow biopsies that have been stained with antibody to von Willebrand factor, which highlights blood vessels. In the left panel, normal bone marrow (from a child with a non-neoplastic disease) shows normal microvasculature of uniform-sized vessels. In the right panel, bone marrow from a child with newly diagnosed acute lymphoblastic leukaemia reveals intense neovascularization, with microvessels of variable diameters. Angiogenesis inhibitors might therefore be useful in treating haematological malignancies. A recent study reported that the retroviral gene transfer of a vector encoding the direct angiogenesis inhibitors angiostatin and endostatin inhibits bone-marrow angiogenesis and tumour growth in a mouse model of leukaemia137. This therapy was shown to directly inhibit endothelial proliferation in vitro, but had no effect on leukaemia-cell proliferation. Mice that were inoculated with B-cell, T-cell or myelogenous leukaemias and treated with recombinant endostatin have also been observed to live significantly longer and experience fewer toxic side effects than with conventional chemotherapy (Timothy Browder et al., unpublished observations). These images are confocal microscopic sections of bone-marrow biopsies that have been stained with antibody to von Willebrand factor, which highlights blood vessels. In the left panel, normal bone marrow (from a child with a non-neoplastic disease) shows normal microvasculature of uniform-sized vessels. In the right panel, bone marrow from a child with newly diagnosed acute lymphoblastic leukaemia reveals intense neovascularization, with microvessels of variable diameters.

matrix metalloproteinase inhibitors MMPIs Phase III clinical trials with MMPIs (Marimastat – British Biotech) in several solid tumours Disappointing results. Reasons may be Early initiation of advanced testing (Phase III) without the appropriate safety and efficacy indications from Phase I/II trials. side-effects (mainly musculoskeletal pain) associated with patient non-compliance in trials. inappropriate model (advanced-stage refractory diseases) in spite of preclinical testing in animal models that had indicated an advantage at an early stage of disease. Poor survival rate in phase III clinical trials against renal cell carcinoma

References 1) Angiogenesis modulation in cancer research:Novel clinical approaches by M Cristofanilli, C Charnsangavej‡ and GN.Hortobagyi Nature reviews drug discovery VOL 1 JUNE pp 415 (2002) 2) Angiogenesis in cancer and other diseases by P Carmeliet & RK. Jain Nature vol 407 14 september 2000 pp 249 3) Chapter 17 : Knowles and Selby