Tumor rim cells: From resistance to vascular targeting agents to complete tumor ablation

Abstract

Current vascular targeting strategies pursue two main goals: anti-angiogenesis agents aim to halt sprouting and the formation of new blood vessels, while vascular disrupting agents along with coaguligands seek to compromise blood circulation in the vessels. The ultimate goal of such therapies is to deprive tumor cells out of oxygen and nutrients long enough to succumb cancer cells to death. Most of vascular targeting agents presented promising therapeutic potential, but the final goal which is cure is rarely achieved. Nevertheless, in both preclinical and clinical settings, tumors tend to grow back, featuring a highly invasive, metastatic, and extremely resistant form. This review highlights the critical significance of tumor rim cells as the main factor, determining therapy success with vascular targeting agents. We present an overview of different single and combination treatments with vascular targeting agents that enable efficient targeting of tumor rim cells and long-lasting tumor cure. Understanding the nature of tumor rim cells, how they establish, how they manage to survive of vascular targeting agents, and how they contribute in tumor refractoriness, may open new avenues to the development of beneficial strategies, capable to eliminate residual rim cells, and enable tumor ablation once and forever.

Keywords

Vascular targeting agents anti-angiogenesis agents vascular disrupting agents coaguligands tumor resistance tumor rim cells complete tumor ablation

Introduction

Mode of action of vascular targeting agents

In recent years, targeting the tumor cell–specific markers is widely applied in oncology practice.^1,2 Although these therapies resulted in beneficial effects for subsets of tumors, the ultimate goal, which is cure, is very rarely accomplished.³ However, the unique characteristics of tumor vasculature confer it an ideal target for the development of selective vascular targeting agents (VTAs).^4–7 Current VTAs split into three main groups, and through a different mode of action, they all aim to cut off tumor nutrient and oxygen path, to starve and suffocate tumor cells to death.⁸

Inhibition of tumor neovascularization by anti-angiogenesis agents

The history of anti-angiogenesis approach goes back to 1971, when Judah Folkman reported the isolation of a pro-angiogenic tumor factor, and the same year recommended the exploitation of anti-angiogenic agents as anti-cancer drugs.⁹ The purpose of anti-angiogenesis therapies is to halt generation of new blood vessels (sprouting)^10–14 or alternatively to inhibit proliferation of tumor endothelial cells (ECs).^15–18 Examples of different classes of these anti-angiogenesis agents (AAAs) are listed in Table 1. While most AAAs are reported to exacerbate hypoxia in tumor cells, anti–vascular endothelial growth factor (VEGF) antibodies, for example, bevacizumab, are reported to improve blood flow by decompression of blood vessels and normalizing tumor vasculature.

Table 1.

Different classes of anti-angiogenesis agents (AAAs).

	Drug type	Drugs
Inhibitors of pro-angiogenic factor secretion	Direct HIF inhibitor	PX-478
	Hsp-90	Radiocol and Geldanamycin
	Thioredoxin	PX-478, Leurotin, and AJM290
	Topoisomerases	Irinotecan, Topotecan, GL331, and Anthracycline
	Microtubule complexes	2-Methoxyestradiol (2-ME)
	Histone deacetylases	Dacinostat
	PI3K	Wortmannin and LY294002
	Guanyl cyclase	NS2028
	mTOR	Everolimus, Temsirolimus (CCI-779), and Sirolimus (Rapamycin)
Ligand-binding agents	VEGF-A	Bevacizumab (Avastin)
	VEGF-A	Bevacizumab (Lucentis)
	VEGF-A	Pegaptanib (Macugen)^a
	VEGFR1 and VEGFR2	VEGF-Trap
	VEGFR2	IMC-1121B
	Platelet-derived growth factor, aFGF, bFGF, VEGF, transforming growth factor-β and insulin-like growth factor-1	Suramin
Agents that target the angiogenic receptors/tyrosine kinase activity	VEGFR2	DC101
	VEGFR and PDGFR	AG-013925
	VEGFR1–3, PDGFR-α, PDGFR-β, c-Kit, CSF-1R, and Flt-3	Sunitinib (SU11248)
	VEGFR2, PDGFR-β, FGFR1, and c-kit	Semaxanib (SU5416)
	VEGFR1–3, PDGFR-β, Raf-1, and B-Raf	Sorafenib
	VEGFR1–2, PDGFR-α, PDGFR-β, and c-kit	SU11657
	VEGFR2, FGFR1, and PDGF-β	SU6668
	VEGFR1–3, PDGFR-β, and c-kit	Vatalanib
	VEGFR2–3 and EGFR	Vandetanib (ZD6474)
	VEGFR1–3, PDGFR-β, and c-kit	ZD2171
	VEGFR1–3, PDGFR-β, and c-kit	Axitinib (AG-013736)
	VEGFR1–3	PTK787/ZK222584
	VEGFRs and FGFRs	Brivanib
	VEGFR2 and EGFR	ZD6474
	VEGFRs, FGFRs, and PDGFR	Nintedanib
	VEGFR2 and EGFR	AEE788
	FGFR1–3	AZD4547
	FGFR1–4	Ponatinib
	FGFRs	SSR
	FEGFRs, VEGFRs, and PDGFR	Dovitinib
	Bcr-Abl, PDGFR-β, and c-kit	Imatinib
	KDR/Flk-1 receptor	VEGF121
Endogenous AAAs	Thrombospondin-1, angiostatin, angiopoietin 2, endostatin, tumstatin, arresten, canstatin, vastatin, and restin IL-12, IL-18, and interferon α Dopamine
Chemotherapeutic agents	Induce EC apoptosis	Cyclophosphamide
	Microtubule	Paclitaxel
	Various	Methotrexate
	Various	Doxorubicin
	COX2	Celecoxib
	Various	Thalidomide
mAb against tumor endothelial–associated antigens/markers	Galectin-1 (gal-1)	Anginex
	α_vβ₃ antagonists/α_vβ₅ antagonists	LM609, 9G2.1.3, RGD, NGR, Vitaxin, CNTO95, EMD121974 (Cilengitide), Volociximab, and Etaracizumab
	CD13/aminopeptidase N (APN)	NGR and Curcumin
	Anti-phosphatidylserine monoclonal antibody	Bavituximab
	Angiotensin II receptor	R6313/G2
	EDB of fibronectin	L19/ L19-SIP
	EDA of fibronectin	F8
	Splice-isoform A1 of tenascin-C	F16
	Endoglin (CD105)	TRC105
	CD44	TES23
	Endosialin (CD248/tumor endothelial marker 1 (TEM1))	Ontuxizumab (MORAb-004)
	MMP	Marimastat/BMS-275291
	CD36	Thrombospondin-1 (ABT-510)
	Inhibits heparin-binding growth factors	Pentosan polysulfate

HIF: hypoxia-inducible factor; mTOR: mechanistic target of rapamycin; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; aFGF: acidic fibroblast growth factor; bFGF: basic fibroblast growth factor; FGFR: fibroblast growth factor receptor; PDGFR: platelet-derived growth factor receptor; CSF-1R: colony stimulating factor 1 receptor; KDR: kinase insert domain receptor; EC: endothelial cell; EDA: extracellular domain A; EDB: extracellular domain B; MMP: matrix metalloproteinase.

RNA aptamer for VEGF-A.

Disruption of tumor blood vessels by vascular disrupting agents

In the early 1980s, Juliana Denekamp verified that tumor regression occurs when the blood flow is compromised.¹⁹ Vascular disrupting agents (VDAs) exploit the distinct shape of tumor ECs to induce selective vascular shut down, without requiring a targeting moiety. There are two types of VDAs: flavonoids, including 5,6-dimethylxanthenone 4-acetic acid (DMXAA) and its synthetic derivatives, and tubulin-binding agents, for example, combretastatin A-4 phosphate, ZD6126, and Oxi4503.²⁰ The mode of action of flavonoids is to cause semi-disintergration of the actin cytoskeleton, division of DNA strands, and induction of EC apoptosis.²¹ However, tubulin-binding agents disturb the ECs’ dependence on the tubulin cytoskeleton to retain their shape.²²

Infarction of tumor blood vessels by coaguligands

Myocardial/brain infarctions are the top leading causes of death around the globe. A single blood clot would be enough for congestion of a single capillary and subsequent death of a thousand cells. The same strategy could be adopted to combat cancer by generation of tumor-specific coaguligands.²³ Coaguligands consist of a truncated form of human tissue factor (tTF), which is fused to a tumor EC moiety. Upon binding to the tumor vasculature, TF induces selective thrombosis, leading to subsequent ischemia, and infarction of tumor blood vessels.^24,25 In contrast to VDA therapy, TF-induced tumor vascular infarction has met with beneficial effects in eradication of large tumors in animal models, yet this approach suffers from incomplete induction of thrombosis, which leaves a ring of viable tumor cells at the tumor edge, similar to the effect of VDAs on large bulky tumors.^26,27

Residual rim cells: the main barrier to successful VTA therapy

The unequal oxygen distribution found within solid tumors results in the establishment of three distinct layers of cells: (1) the anoxic center core composed of necrotic cells, (2) the hypoxic middle layer harboring dormant cancer cells, and (3) the normoxic outer layer featured by highly proliferative tumor cells (Figure 1).²⁸ The outer proliferative layer is a target for tumor-reducing agents, including chemotherapy and anti-angiogenesis, which elicit cytostatic effects on the highly proliferating cells at the tumor periphery.²⁹ However, the necrotic inner layer provides a heaven for localization of anaerobic bacteria.³⁰ The interstitial locations of cells in the middle layer, however, render them out of reach of most anti-cancer agents. More importantly, constant low-grade hypoxia in the middle layer selects for hypoxia-tolerant cells, known as tumor rim cells, extremely resistant cells to eradicate.²⁸ The most effective VDAs producing more than 90% widespread central necrosis still leave a thin ring of viable cells from which tumor revives.²⁰ It appears that the existence of tumor rim cells remains the foremost impediment to successful tumor ablation encountered by vascular targeting strategies, especially VDAs.³¹ Although there is no evidence for the existence of tumor rim cells following anti-angiogenic treatment, tumor survival and aggressive phenotype encountering by AAAs justify the likelihood of the existence of such resistant population of tumor cells. The running hypothesis for the ineffectiveness of VDAs to eradicate tumor rim cells is that rim cells might nourish from surrounding normal vasculature; hence, they are immune to vascular disruption and hypoxia.³ Although, potent VDAs are shown to induce widespread hypoxia, encompassing viable tumor edge cells, as well.²⁰ Furthermore, it is established that widespread thrombosis induced by TF fusion proteins leads to tumor vascular infarction, ischemia, and degeneration of tumor cells, as well as the tumor rim cells and cure.^23,32 These observations suggest that hypoxia-tolerant tumor rim cells are not ordinary tumor cells to target, yet sustained deprivation of tumors out of oxygen and nutrients would suffice to succumb these tough cells to death.³² As most VTAs are shown to induce a high grade of hypoxia, rather than anoxia, tumors are forced to divert to compensatory mechanisms, including switching to alternative angiogenic pathways,^33,34 stimulating dormant cells,³⁵ selecting aggressive and metastatic cells,^36,37 autophagy,³⁸ establishment of cancer stem cells,^39,40 and recruiting vascular progenitor cells^41,42 as well as tumor-associated macrophage (TAM).^43–45 Features that tumor exploits to escape oddly shape the typical characteristics of challenging tumor rim cells as well.²⁸

Figure 1.

The characteristics of different layers of tumors. Spontaneous solid tumors are composed of three distinct layers of cells. The outer proliferative layer which is in close contact to the bloodstream and could be readily targeted by most anti-cancer drugs. The necrotic inner layer (tumor core) provides a heaven for propagation of anaerobic bacteria that feed on tumor cells/or alternatively deliver cytotoxic drugs to the tumor core. The interstitial location of cells in the middle layer, however, renders tumor cells out of reach of cytotoxic drugs that could attack tumor cells either from inside (tumor core) or outside (the tumor periphery). Furthermore, adaptation of tumor cells to the lower O₂ tension in the middle layer selects for the dormant aggressive cells–the rim cells—hard to target and eradicate by current anti-cancer approaches. Rim cells occasionally could be targeted by coaguligands. Smart combination rational, however, could increase the chance of targeting rim cells and enable tumor cure.

Eradication of tumor rim cells and complete remission of tumor by single/combination therapies with VTAs

Eradication of tumor rim cells by VTAs (single modalities)

Among different vascular targeting strategies, several approaches reported to elicit curative potentials as single therapies in preclinical settings. Complete tumor ablation by overcoming tumor rim cells is accomplished with ligand-directed vascular targeting, coaguligands, DNA vaccines, and photodynamic therapy (PDT; Table 2).

Table 2.

Complete tumor ablation by single/combination therapy with VTAs.

No.	Cell line(s)	Drugs and doses	Tumor type	TV before treatment (mm³)	CR (%)	Year	Ref.
1	FaDu	Bevacizumab (5 mg/kg/daily × 28 days, i.p.) + Irinotecan (100 mg/kg, weekly × 4, i.v.)	Head and neck squamous cell carcinoma (HNSCC)	200–250	~80% (7/9)	2011	Cao et al.⁴⁶
2	MA148	Rofulven (q3dx4) i.p., + Anginex/0118 (10 mg/ kg/day × 28 days, s.c.)	Ovary	100	100% (6/6)	2008	Dings et al.⁴⁷
3	MDA-MB-231	4/8 mg/kg bevacizumab twice a week, i.p. + 10 mg/kg nab/paclitaxel qdx5 repeated 2–3 times with 1 week of rest interval, i.v.	Breast	250–310	40% (2/5) and 80% (4/5)	2008	Volk et al.⁴⁸
3	MCF-7	20 µg/200 µL mAb–dgRA (q2 × 2, i.v.)	Breast	Impalpable	100% (8/8)	1993	Burrows et al.⁴⁹
4	MCF-7	SN6j-dgRA or SN6k-dgRA (40 µg q2d × 2, i.v.)	Breast	40–90	~65% (5/8)	1997	Seon et al.⁵⁰
5	C1300 (Muў)/	11-4.1-dgA (100 µg, i.v.) + M5/114 dgA (40 µg, i.v.)	Neuroblastoma	0.8–1.2 cm	~65% (5/8)	1999	Matsuno et al.⁵¹
6	Ramos, DoHH-2	200 µg rituximab + 20 µg L19-IL2, i.v. (q3d × 4)	Lymphoma	50–100	Ramos: 80% (4/5) and DoHH-2: 100% (5/5)	2008	Schliemann et al.⁵²
7	CT26, F9	30 µg L19–IL2 (given 24 before or after) +50 µg of anti-CTLA-4 antibody (q4 × 2 F9) q3d × 3 CT26), i.v. 30 µg L19–IL2 and 7 µg L19-TNF (×2), i.v.	Colorectal and Teratocarcinoma	70, 150, and210	F9: 30% (3/10); CT26: small tumors 100% (9/9) and large tumors 60% (3/5). F9: small tumor 100% (5/5) and large tumors ~85% (5/6)	2012	Schwager et al.⁵³
8	C1498	F8-IL2 (1 mg/kg; days 7, 10, and 13) + F8-SS-CH2Cem (10 mg/kg; days 8, 9, 11, and 12), i.v.	Acute myelogenous leukemia (AML)	50–100	80% (4/5)	2014	Gutbrodt et al.⁵⁴
9	F9	IL-12–L19, and TNF-α–L19 (2 µg + 2 µg/mouse) × 2	Teratocarcinoma	300	F9: 80% (4/5) and 75% (3/4)	2003	Halin et al.^55,56
10	CT26, A20, and Wehi-164	90 µg F8-IL4+20 µg F8-IL2+8.75 µg IL12-F8-F8 q48h × 4 (i.v.)	Colorectal Lymphoma and fibrosarcoma	70	F9:80%(4/5), CT26: 40% (2/5), and A20: 100% (5/5)	2013	Hemmerle et al.⁵⁷
11	F9	150 µg F8-IL13 + 8.75 IL12-F8F8	Teratocarcinoma	~100	80% (4/5)	2015	Hess et al.⁵⁸
12	CT26, A20, F9	Paclitaxel (10 mg/kg, starting on day 4, q96 h × 2) + mIL12-F8-F8 (8.75 mg, starting on day 5, q 48 hours × 4 times, i.v.	Colorectal, lymphomas, and teratocarcinoma	50	F9 50% (2/4) CT26:40% 2/5 A20: 100% (5/5)	2012	Pasche et al.^59–61
13	E.G7	80 µg OVA DNA + inoculation with 1 × 10⁶ Pan-IA-loaded. Vaccination was repeated four times weekly (i.v.)	Lymphoma	4–5 mm in diameter	70% (14//20)	2007	Teramoto et al.⁶²
14	U87MG	0.525 mg temozolomide, i.p. (q3d × 4) 20 µg F16–IL2 (i.v.)	Glioma	300	100% (5/5)	2013	Pedretti et al.⁶³
15	GEO	ZD6474 (100 or 150 mg/kg/dose) i.p. on days 1–5 of each week for 4 weeks + Paclitaxel (20 mg/kg/dose) on day 1 of each week for 4 weeks.	Colon	200–300	40% (6/20)	2003	Ciardiello et al.⁶⁴
16	MCF-7 and HT29	Dopamine (50 mg/kg/day (i.p.) × 7 continuous days), doxorubicin (5 mg/kg/i.p./single dose), and dopamine (50 mg/kg/day (i.p.) × 7 continuous days) + doxorubicin (5 mg/kg/i.p./single dose). Dopamine (50 mg/kg/day i.p. × 7 continuous days), 5-FU (20 mg/kg i.p. for 5 continuous days), and dopamine (50 mg/kg/day, i.p. × 7 continuous days) + 5-FU (20 mg/kg i.p. for 5 continuous days	Breast and colon	100	~15% (2/14) ~22% (3/14)	2008	Sarkar et al.⁶⁵
17	MCF-7	Suramin (10 mg/kg) + paclitaxel (15 mg/kg, i.v.) twice weekly for 2–3 weeks.	Breast	5–10 mm in diameter	63.2% (19) 50% (6)	2004	Song et al.⁶⁶
18	F9 and A431	SIP (L19)–PS (150 µg, i.v.), followed by 3 min irradiation (60 J cm−²) 24 h later.	Teratocarcinoma	F9 (~50) A431 (50–100)	F9: 75% (3/4) A4311: 100% (4/4)	2011	Palumbo et al.⁶⁷
19	LS174T	¹³¹I-labled A5B7 (18.5 MBq/50 µg, i.v.) + DMXAA (27.5 mg/kg/i.p.) 48 h after	Colorectal	0.1–0.2	84% (5/6)	1996	Pedley et al.⁶⁸
20	SW1222	7.5 MBq/40 µg ¹³¹I-labeled A5B7 i.v. plus a single 200 mg/kg dose of CA4P i.p. 48 h after	Colorectal	0.1–0.2	84% (5/6)	2001	Pedley et al.⁶⁹
21	MDAH-MCa-4	Paclitaxel (31.6 Lmol/kg/i.p.) was given 4 h before/1 h after DMXAA (80 Lmol/kg/i.p.)	Breast	10–11 mm (0.6 g)	43% (3/7)	2003	Siim et al.⁷⁰
22	MDA-MB-435	Single i.v. injection of ZD6126 (200 mg/kg i.p.) administered 72 h before PTX (40 mg/kg i.v. × 3q7d)	Breast	450 mg	57% (4/7)	2007	Martinelli et al.⁷¹
23	DU145	Doses of docetaxel (i.v.) 5 mg/kg with DMXAA 21 mg/kg × 2q4d	Prostate	–	43%	2005	Green et al.⁷²
24	NCI H460, A549, DU145, and SKOV-3	Paclitaxel + DMXAA	Prostate, ovary, and lung	–	–	2006	Green et al.⁷³
25	HCT116	5 × 10⁷ Clostridium novyi-NT spores (time 0, i.v.), D10 (0.3 mg/kg/i.v.) at 24 h, and MMC (4 mg/kg/i.p.) at 48 h.	Colon	700	50% (4/8)	2001	Dang et al.⁷⁴
26	DLD1, SW480, LS174T, HT29, HCT116-p53−/−, HuCC-T1, CaPan-1, and B16	Single i.v. injections of MAC321 (40–70 mg/kg)/Docetaxel (50–70 mg/kg)/vinorelbine (20 mg/kg)/HTI-286 (1.7–2 mg/kg) followed by i.v. injection of 30 × 10⁷ C. novyi-NT spores	Colorectal, cholangiocarcinoma, pancreatic, and melanoma	150–500	>40%	2004	Dang et al.⁷⁵
27	EL-4 and LLC	Injection of cDNA (60–180 µg, i.v.) encoding B7.1, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg)	Lymphoma	0.8 cm	–	2001	Kanwar et al.⁷⁶
28	tTF-B21-2/10H1O 0.33 µg Ab and 0.17 µg tTF (q6day × 1, i.v.)	Nouroblastoma C1300 (Muγ)	0.8–1.0 cm	BALB/c/nude mice	38%	1997	Huang et al.^32,77,78
29	tTF-L19 35 µg q75h × 3, i.v.	FE8 sarcoma	~500 mm³	BALB/c/nude mice	30%	2001	Nilsson et al.⁷⁹
30	0.1 mg/kg tTF-PSMA + 2 mg/kg liposomal doxorubicin (q2 × 2, i.v.)	Human LuCap prostate tumor	200 mm³	Rat	–	2002	Liu et al.⁸⁰
31	tTF-anti VEGF 20 µg × 2q1w i.v.	CT26 colon carcinoma	6–8 mm	Balb/c mice	20% (2/10)	2008	Huang et al.^32,77,78
32	tTF-NGR 3–5 mg/kg q2d × 4, s.c.	HT1080 fibrosarcoma	300–1700 mm³	BALB/c/nude mice	~15% (1/7)	2009	Bieker et al.⁸¹

TV: tumor volume; i.p.: intraperitoneal; i.v.: intravenous; s.c.: subcutaneous; DMXAA: 5,6-dimethylxanthenone 4-acetic acid; MMC: mitomycin-C; cDNA: complementary DNA; FAA: flavone-8-acetic acid; A5B7: anti-carcinoembryonic antigen IgG; CR: complete remission; q: interval between injections; “×”: number of repeated doses; PSMA: prostate-specific membrane antigen; tTF: truncated form of human tissue factor.

Ligand-directed vascular targeting

As a potent anti-angiogenic strategy, ligand-directed vascular targeting is recognized as a worthy anti-cancer approach. For this, ligand could be a monoclonal antibody (mAb)/molecule against a proliferation-associated antigen on ECs and the VTA could be a toxin (immunotoxin (IT)),^{50,51,82–84} cytokine (immunocytokine (IC)),^{52–61,63,85–89} apoptotic, cytotoxic (antibody–drug conjugate (ADC)),^54,90,91 photosensitizer (PS),^92,93 radioisotopes (radionuclides and radiotracers),^94–96 or coagulation factor (coaguligand).^{23,32,77–81,97} Examples of different ligand-directed vascular targeting are listed in Table 3. When targeted to tumor vasculature, ligand-directed VTA inhibits tumor-associated angiogenesis and destroys tumor vasculature by selectively reacting with proliferating ECs.⁵⁴ However, activated/proliferating ECs facilitates endocytosis of the fusion protein inside tumor cells, where the internalized payloads could lead to further cytotoxicity. Matsuno et al.⁵¹ developed anti-endoglin mAbs conjugated to the deglycosylated ricin A (dgRA) chain and tested their anti-tumor efficacy for treating MCF-7 breast carcinoma xenografts in mice. Long-lasting complete regression of the tumors was induced, when three doses of 40 µg of the ITs were administered. In a similar study by Seon et al.,⁵⁰ long-lasting complete remission (CR) was accomplished in all animals (100%), which were given three doses of mAb-dgRA.

Table 3.

Different ligand-directed VTAs.

No.	Drug type	Drug	Ref.
1	Photosensitizer	SnChe₆-(SIP)L19	Birchler et al.⁹² and Fabbrini et al.⁹³
2	Coagulation factor	tTF-RGD	Kessler et al.⁹⁷
		tTF-NGR	Bieker et al.⁸¹
		tTF-L19	Nilsson et al.⁷⁹
		tTF-VCAM-1	Ran et al.²³
		tTF-B21-2/10H1O	Huang et al.³²
		tTF-anti-VEGF	Huang et al.⁷⁷
		(RGD)3-tTF	Huang et al.⁷⁸
		tTF-PSMA	Liu et al.⁸⁰
3	Radionuclide	¹³¹I-L19 SIP ¹²⁵I-L19-SIP ¹⁷⁷Lu-L19-SIP	Tijink et al.⁹⁵
		¹²⁵Iodine-scFv (L19)	Demartis et al.⁹⁴
		²¹¹Astatine-scFv (L19)	Demartis et al.⁹⁴
4	Radiotracers	[18F] Galacto-RGD (2-[18F] fluoropropanamide c(RGDfK(SAA))) [18F]-AH111585	Liu⁹⁶
5	Cytotoxic agents antibody–drug conjugates (ADC)	Doxorubicin–ILA5*	Volkel et al.⁹¹
		Anti-endosialin-MC-VC-PABC-MMAE	Rouleau et al.⁹⁰
		Cemadotin thiol-F8	Gutbrodt et al.⁵⁴
6	Toxins (immunotoxins)	DT (diphtheria toxin)-VEGF	Arora et al.⁸²
		Ricin-A-MHCII	Thorpe et al.^8,83
		SN6j-dgRA SN6k-dgRA	Matsuno et al.⁵¹
		SN6f-dgRA	Seon et al.⁵⁰
		rGel (Gelonin)-VEGF121	Veenendaal et al.⁸⁴
7	Cytokines (immunocytokines)	TNF-α-NGR	Borsi et al.⁸⁵
		GM-CSF-L19	Kaspar et al.⁸⁹
		IL15-L19	Kaspar et al.⁸⁹
		INF γ-L19	Ebbinghaus et al.⁸⁶
		INF α-F8	Frey et al.^87,88
		TNFα-L19	Halin et al.^55,56
		IL2-L19	Schliemann et al.⁵²
		IL12-L19	Halin et al.^55,56
		IL12-F8-F8	Pasche et al.^59–61
		IL2-F16	Pedretti et al.⁶³
		IL2-F8	Frey et al.^87,88 and Gutbrodt et al.⁵⁴
		IL10-F8	Schwager et al.⁵³
		IL7-F8	Pasche et al.^59–61
		IL17-F8	Pasche et al.^59–61
		IL-13-F8	Hess and Neri⁵⁸
		IL4-F8	Hemmerle et al.⁵⁷

MMAE: monomethyl auristatin E; tTF: truncated form of human tissue factor.

SAA: 7-amino-l-glycero-l-galacto-2,6-anhydro-7-deoxyheptanamide.

Tumor vascular infarction

Induction of selective thrombosis in tumor vasculature by TF fusion proteins (coaguligands) leads to severe ischemia, and in case of the maintained complete thrombosis, it could lead to the eradication of tumor rim cells and cure. Huang et al.³² generated the first coaguligand to induce selective tumor vascular infarction in neuroblastoma tumors in mice. A bispecific Ab composed of tTF and Ab against MHC-II—an artificial marker of angiogenesis—was injected into mice bearing large C1300 (Muy) tumors. Upon a single injection, massive thrombosis occurred throughout tumor in 30 min, and extensive intravascular thrombosis of the tumor lasted for more than 72 h, resulting in complete regressions in 38% of the treated mice, lasting 4 months or more. Partial tumor regression in mice was consistent with undetectable MHCII antigens in the tumor periphery and incomplete induction of thrombosis by the coaguligand, which allowed rim cell survival. Nilsson et al.⁷⁹ targeted tTF to EDB domain of fibronectin on vasculature of three different types of solid tumors. Although a single 20 µg injection of the ScFv (L19)-tTF induced the rapid infarction of large tumors, this dose of coaguligand failed to eradicate tumor rim cells, and tumor tends to grow from a residual ring of viable cells close to the peritoneum. Higher doses of coaguligand (35 µg) enabled complete ablation of residual tumor mass and lead to CR in ~30% of the treated mice. Eradication of tumors by other coaguligands, including Ttf–prostate-specific membrane antigen (PSMA), tTF-VEGF, and tTF-NGR, is reported by other groups as well.^77,80,81

DNA vaccines

Given the fact that tumors can continuously undergo mutations to alter their antigenic features, they can evade from host immune responses and become resistant to the anti-cancer therapies.^76,98 Tumors are immunosuppressive in a variety of ways, and pro-angiogenic cytokines and their receptors, including VEGF family, fibroblast growth factor (FGF), and matrix metalloproteinases (MMPs), elicit central role in these immunosuppressive events.^99,100 For example, VEGF may functionally inhibit the immune system, partly by preventing the maturation of dendritic cells and inhibiting early T-cell development.¹⁰¹ In this regard, immune-based cancer therapies that target angiogenesis may potentiate a broader anti-tumor immune responses in various ways: (a) hamper angiogenesis via blocking pro-angiogenic cytokines; (b) ameliorate the tumor immunity by interfering with immunosuppressants; (c) target multiple angiogenic mediators, which enhances the effectiveness of anti-angiogenesis; (d) circumvent the need for repeated drug administration; and (e) moreover, immunotherapy could exert not only therapeutic but also prophylactic potential by suppression/eradication of metastasis.^102–105 In the study by Teramoto et al.,⁶² pigeon cytochrome c–derived peptides (Pan-IA) containing dendritic cells were immunized in combination with ovalbumin (OVA) antigen DNA to prime both T helper and cytotoxic T lymphocyte (CTL) immunity in mice bearing tumor xenografts. Pan-IA is shown to trigger T helper function in mice, through efficient binding to a wide variety of MHC class II molecules. In this study, tumor-specific cytotoxicity and helper immunity were only primed in the combination setting and resulted in augmented tumor lysis ability, interferon (IFN)-gamma-mediated anti-angiogenic effects, and CR of tumor in ~70% of the treated mice.

PDT

PDT is now largely appreciated in treatment of tumors, especially the superficial ones. This modality incorporates a visible light to convert nontoxic oxygen molecules within tumors into cytotoxic singlet oxygen, through a PS. The antibody–PS conjugate could be employed for selective directing of PS to the tumor site.⁹³ PDT consumes tumor oxygen supply to generate reactive oxygen molecules (ROS) and to induce apoptosis/necrosis of tumor cells. In addition, PDT could be formulated to destroy tumor vasculature as well.¹⁰⁶ Palumbo et al.⁶⁷ verified the efficacy of PS-L19 for selective targeting of tumor neovasculature in mice bearing F9 murine teratocarcinoma and A431 human epidermoid carcinoma xenografts. L19 motif allowed specific localization of PS on tumor vasculature in both tumor models in mice. Upon irradiation, porphyrin-based PS promoted PDT and permitted selective tumor vascular disruption, leading to complete and long-term tumor eradication. Furthermore, depletion studies indicated that natural killer (NK) cells were indispensable for lifelong complete responses.

As single modality, a perfect VTA should be able to penetrate deep into the hypoxic regions, where dormant rim cells reside. In addition, such therapy should provide a sustained toxic dose of VTAs enough to kill any residual cancer cells. While most VTAs render a narrow therapeutic dose and fail to provide a higher retention time, combination therapies are indicated to overcome VTA-associated resistance in tumors.

Eradication of tumor rim cells by VTAs (combination therapies)

Most of current VTAs in the clinic need to couple with other anti-cancer modalities; the concerted work of two different agents leads to the enhanced therapeutic potential and enable tumor remission. The aim of such combination therapy is to improve the odds of successful targeting of the rim cells, which lower the risk of subsequent tumor survival and invasion and consequently enable tumor eradication for good. Notably, drug timing and sequencing is of critical significance determining the curative potential for combination therapies with VTAs.

AAAs combined with AAAs/chemotherapy

Given to the redundancy in angiogenic factors, the concept for concurrent application of two different AAAs is clear. Nonetheless, the rational for combination of AAAs with chemotherapy is to improve delivery of chemotherapeutic agents, as the first class of AAAs is shown to improve tumor blood flow by decompression of tumor blood vessels; hence, administration of AAAs prior to intravenously (i.v.) administered chemotherapeutic agents would lead to improved uptake of chemotherapeutic agents by tumor cells. Cao et al.⁴⁶ evaluated the anti-tumor activity of bevacizumab combined with topoisomerase I inhibitor irinotecan (CPT-11) against human head and neck squamous cell carcinoma (HNSCC) xenografts in mice. Daily bevacizumab therapy was initiated 1 week in advance to CPT-11 treatment and continued for 4 weeks. Monotherapy with irinotecan resulted in almost 40% CR, while bevacizumab alone showed no curative potential. Combination therapy of bevacizumab with irinotecan produced marked inhibition of FaDu tumor growth and resulted in 80% CR of tumor in mice. Drug scheduling was found crucial for effective chemotherapeutic effects since bevacizumab potentiated irinotecan curative potential, only when lower doses of bevacizumab administered 7 days prior to irinotecan. Concurrent administration of both drugs and/or injection of higher doses of bevacizumab failed to potentiate anti-tumor activity of irinotecan. In another study, Dings et al.⁴⁷ reported combination of anginex (angiostatic agent) and 0118 (topomimetic of anginex) with chemotherapeutic irofulven to treat human ovarian tumor xenografts in mice. The combination of a low dose of irofulven with either AAAs was more effective at inhibiting tumor growth than any of the monotherapies, as the anginex/irofulven and 0118/irofulven combinations produced 92% and 96% tumor growth inhibition, respectively. The 0118/irofulven combination resulted in 100% complete responses in treated mice. A combination of AAAs with low-dose irofulven not only augmented the anti-tumor efficacy of irofulven but also abolished administration of toxic doses of this chemotherapeutic in single therapy. In an additional study, Volk et al.⁴⁸ explored the anti-tumor and anti-metastatic potential of bevacizumab combined with nab-paclitaxel (an albumin-bound form of paclitaxel) in mice bearing orthotopic breast tumors. Mice bearing luciferase-tagged MDA-MB-231 tumors were treated with one, two, or three cycles of bevacizumab, nab-paclitaxel, or combination of both. Bevacizumab therapy abrogated the VEGF-A protective effect upon nab-paclitaxel cytotoxicity and restored breast tumor sensitivity to chemotherapy. Combined work AAA and chemotherapeutic nab-paclitaxel markedly inhibited tumor growth and metastasis, resulting in up to 40% CR. This therapy was also promising in reduction of both lymphatic and pulmonary metastases by 60% and 100%, respectively. Burrows et al.⁴⁹ examined the single and combined therapeutic potential of EC IT (anti MHCII) M5/114-dgA and anti-tumor IT 11-4.1-dgA (anti MHCI) in large neuroblastoma tumors. In a dose-dependent fashion, low doses of M5/114-dgA (20 µg) gave negligible and transitory responses in animals, whereas high doses (40 µg) of IT resulted in marked regression of tumors in all animals, but the responses were temporary, since tumors started to repopulate 1 week after. A combination of two ITs led to long-term eradication of tumors in 5/8 of the animals. In view of the fact that the efficacy of rituximab-based therapies is largely relied upon the extent of ADCC-mediated destroying of lymphoma cells, Schliemann et al. adopted different single and combination treatment schedules of rituximab (mAb against CD20 on the surface of B cells) and L19-IL2 in CB17/lcr SCID mice bearing Ramos and DoHH-2 human B-cell lymphoma xenografts. In Ramos tumor xenografts, CR of tumor (4/5 mice) was observed, when 200 µg rituximab combined with 6.6 or 20 µg L19-IL2; however, long-lasting CR for more than 3 months was observed with higher doses of L19-IL2 (4/5 mice). The results were reproducible, and in DoHH-2 follicular lymphoma xenografts, 200 µg rituximab + 20 µg L19-IL2 led to additive effects and long-lasting CR in all animals (5/5 mice). Gutbrodt et al.⁵⁴ treated immunocompetent mice bearing acute myelogenous leukemia (AML) tumors with a combination of cemadotin thiol or IL2 as cytotoxic drugs containing F8 as a vascular targeting motif. Anti-hen egg lysozyme Ab KSF was served as a negative control. When tumors reached to the volume of 50–100 mm³, mice were given i.v. injections of either saline, F8-IL2 (1 mg/kg), F8-SSCH2Cem (10 mg/kg), KSF-SS-CH2Cem (10 mg/kg), or F8-IL2 + F8-SS-CH2Cem. Combination of F8-SS-CH2Cem and F8-IL2 resulted in long-lasting complete tumor eradication in 4/5 mice. Furthermore, upon rechallengement with C1498 cells after ~4 months, all cured animals rejected tumors, indicating the acquired protective immunity in this setting. Schwager et al.⁵³ evaluated the efficacy of L19–IL2 in combination with a murine anti-CTLA-4 antibody (a mouse analog of Ipilimumab) or with L19-TNF in F9 murine teratocarcinomas and CT26 colon carcinoma xenografts. CR was demonstrated in both tumor models, when L19–IL2 was combined with CTLA-4 blockade. Since in F9 tumors the CTLA-4 blockage is not active and NK cells are the main regulators of therapeutic activity of targeted IL2, cured mice from F9 tumors developed lesions upon rechallenging with F9 tumor cells. In the CT26 model, T-cells are the main mediators accounting for anti-tumor action of IL2 and cured mice remained tumor free upon rechallenging with the parental cancer cells. Similarly, a single intratumoral injection of L19–IL2 induced CR once it coupled with L19-TNF therapy, whereas the two drugs failed to produce any cures as single modalities. Pasche et al.⁵⁹ explored therapeutic performance of IL-12 combined with IL2 and paclitaxel. In this study, the combination of F8-IL2 and mIL12-F8-F8 led to anti-angiogenic effects induced by activation of INFγ and CR in 2 of 3 mice bearing F9 tumors. This was due to the intratumoral administration of the ICs, but only at the cost of excessive toxicity. While single high dose (10 mg/kg) of paclitaxel conferred just a slight tumor growth retardation, the combination of low-dose paclitaxel (8.75 mg) with mIL12-F8-F8 led to a maintained CR in 2/4 mice bearing F9 tumors. Similar successful therapies reporting eradication of tumors by the combination of AAAs with AAAs/chemotherapeutic agents are listed in Table 3.^{55,57,58,63–66}

VDAs combined with chemotherapy

Combination of VDAs which confer the best effects on bulky tumors with chemotherapeutic agents should be given in an opposite order to that of AAAs. Here, chemotherapeutic agents should be administered in advance to the chemotherapeutics. Such combination would lead to the occlusion of blood vessels, which entrap chemotherapeutic agents, and as a result increase retention of chemotherapeutics. In addition, VDA therapy following chemotherapy may avoid rapid clearance of chemotherapeutic drugs from the bloodstream; consequently, the therapeutic efficacy improves. However, the reverse order of drugs may produce similar beneficial effects. VDA therapy given before/along with chemotherapy leads to the activation/proliferation of dormant rim cells, which render them susceptible to the chemotherapeutic agents. The critical impact of drug schedule is clearly reflected in the study conducted by Martinelli et al.⁷¹ In this study, authors examined the impact of schedule and sequence on combined therapeutic potential of a microtubule destabilizing VDA (ZD6126) and a microtubule stabilizing chemotherapeutic paclitaxel in mice bearing MDA-MB-435 breast tumor xenografts. Weekly cycles of ZD6126 (200 mg/kg intraperitoneal (i.p.)) were injected at different time points prior or after paclitaxel (10, 20, and 40 mg/kg i.v.). Protective effect of paclitaxel toward ZD6126 failed to produce any measurable benefit, when VDA administered 2 or 24 h after chemotherapy. Distancing drug administration by 72 h counteracted the opposing effects of paclitaxel and boosted the anti-tumor potential of VDA therapy. Furthermore, schedules in which VDA were given before chemotherapeutic were consistent with VDA-induced proliferation of tumor rim cells, leading to additive cytotoxicity. The most effective response was accomplished when VDA administered 72 h before paclitaxel (50% CR). A single injection of VDA followed by weekly chemotherapy yielded a similar response (57% CR). In another study, Siim et al.⁷⁰ verified the anti-tumor effects of DMXAA combined with several chemotherapeutic agents in murine mammary carcinoma xenografts. The observed additive effects in combination of VDA with different chemotherapeutics were in the order of 5-fluorouracil < (etoposide, carboplatin, cyclophosphamide, doxorubicin, cisplatin) < (docetaxel, vincristine) < paclitaxel. The interaction of DMXAA with paclitaxel was prominent and the combined therapy resulted in extension of the median tumor growth delay from 0.3 to 80 days, with 3 of 7 mice cured. This combination showed a broader timing, as paclitaxel produced similar activity when administered 4 h before to 1 h after VDA. Notably, VDA administration 4 h before paclitaxel conferred no cures. Green et al.^72,73 demonstrated synergism effects associated with tumor cures, when DMXAA was combined with either paclitaxel (in non-small-cell lung carcinoma (NSCLC) lung and ovarian models) or docetaxel (in prostate model).

VDAs combined with immunotherapy

Immunotherapy is curative against small tumors in animal models of cancer, the foremost obstacle that must be addressed relates to the immune resistance of large tumors. This immune resistance could be surmounted by combining immunotherapy with a rigorous attack of VDAs on the vasculature of large tumors, leading to finely tuned immune responses, potentiating tumor ablation, and more importantly, eliciting anti-tumor immunity.¹⁰⁷ For this, Kanwar et al.⁷⁶ combined DMXAA and flavone acetic acid (FAA) with B7.1 (CD80) to trigger anti-tumor immunity in mice bearing large lymphoma xenografts. B7.1 is known to costimulates proliferation of T-cells through CD28 pathway. B7.1 complementary DNA (cDNA) was injected into EL-4 tumors and 24 h later followed by VDA therapy of either DMXAA or FAA. Employing this schedule, tumors were successfully removed within 2–6 weeks. CD8+ T-cells and NK cells were active in the combination setting, which resulted in heightened and sustained anti-tumor cytolytic activity and enhanced destruction of cells by inducing apoptosis. This combination therapy resulted in acquired tumor-specific immunity in cured animals, since treated mice were immune to a challenge of 1 × 10⁷ parental lymphoma cells but not a challenge of 1 × 10⁴ Lewis lung carcinoma cells. Moreover, adoptive transfer of 2 × 10⁸ splenocytes from cured animals into the tumor bearing recipients prompted fast and full rejection of tumors within 3 weeks.

VDAs combined with anaerobic bacteria

Anaerobic bacteria are capable to selectively localize into the avascular necrotic regions of tumors which are normally inaccessible to intravenously administered drugs.¹⁰⁸ However, the low oxygen tension in this area confers these cells less susceptible to radiation and chemotherapeutic agents.^109,110 Albeit, there are reports regarding successful treatment of established tumors by single-bacteria therapy,^111–114 monotherapy with bacteria is limited mostly due to the concerns regarding bacteria toxicity and incomplete tumor lysis.¹⁰⁸ Since the hypoxia tension varies in different parts of the tumor, bacteria cannot consume all parts of the malignant tissue. For this, combination therapy with chemotherapeutic treatments (COBALT) may resolve the problem by offering synergic effects. Such combination may employ small molecules to kill cancer cells close to blood vessels, while bacteria could destroy the cells in the hypoxic niche, located far from tumor nourishing vessels.²⁸ Dang et al.⁷⁴ verified the therapeutic potential of COBALT in combination with VDAs in HCT116 colorectal cancer and B16 melanoma xenografts. Their rational was that VDAs can trap bacteria spores within avascular regions of tumors, buying time for the spores to germinate. Moreover, the vascular collapse would lower the oxygen tension close to the trapped bacteria and thereby increases the hypoxic region for bacterial growth in tumors. In addition, proliferating tumor rim cells could be killed upon exposure to the chemotherapeutic agent. To this end, 26 bacterial strains and 9 VDAs were screened to find the best candidates for combination therapy. Clostridium novyi-NT was found the best candidate due to its capability for extensive spreading throughout avascular regions of the tumors and for devoiding lethal toxins. Animals were given i.v. doses of 5 × 10⁷ C. novyi-NT spores (time 0), followed by i.v. injection with D10 as VDA (0.3 mg/kg) at 24 h and i.p. injection with chemotherapeutic agent MMC (4 mg/kg) at 48 h. Full eradication of large tumors was observed in 50% of mice. Unfortunately, the striking anti-tumor effects of COBALT were attended with severe toxicity probably due to lysis syndrome. In another study, Dang et al.⁷⁵ explored the therapeutic performance of C. novyi-NT combined with VDAs (HTI-286 and vinorelbine) and vascular stabilizing agents (taxanes such as the docetaxel and MAC321). Results indicated that VDAs but not taxanes compromised the blood flow to tumors, which led to enlarging the hypoxic region, where germination C. novyi-NT spores could efficiently proceed. Combination of C. novyi-NT (a single injection) with VDAs was able to ablate various tumor xenografts without severe toxicity.

VDAs combined with radioimmunotherapy

The successful targeting of radio-labeled antibodies to tumor sites was directed to the advent of radioimmunotherapy (RIT). Beta particle isotopes, including ¹³¹I and ⁹⁰Y, could emit their energy throughout a large portion of tumor. RIT is proved as a beneficial modality in cancer therapy since there is no need for drug internalization or binding to each individual cell to elicit cytotoxicity.^34,115 VDA administration 48 h before RIT could enhance antibody tumor localization and retention and enable removal of any residual viable cells upon irradiation.^68,69 Pedley et al.⁶⁸ explored the efficacy of RIT plus DMXAA in LS174T colorectal carcinoma xenografts. A single administration of DMXAA (27.5 mg/kg) failed to clear viable rim cells; however, when the same dosage was coupled with 18.5 MBq/50 µg ¹³¹I-labled anti-carcinoembryonic IgG, prolonged CR was accomplished in five of six mice. In another study, Pedley et al.⁶⁹ evaluated the efficacy of CA4P combined with RIT in SW1222 xenografts. Upon monotherapy, a narrow viable cells survived at the tumor edge, while in combination setting, 7.4 MBq ¹³¹I-labeled anti-carcinoembryonic antigen IgG plus a single 200 mg/kg dose of CA4P produced complete cures in five of six mice lasting for >9 months.

Conclusion and future direction

Vascular targeting approaches as single modalities or in combination with other anti-cancer strategies demonstrated beneficial effects in inhibition/eradication of established tumors in mice. The main impediment to complete cure, at least when a VTA is implicated, is the existence of viable residual cells called as tumor rim cells; because of their special characteristic, tumor rim cells are inaccessible and less susceptible to most anti-cancer drugs. In most cases, implementation of long-lasting toxic doses of anti-cancer agents is required to eradicate tumors in single modalities, and since these two key features (effective dose and high retention time) are beyond our grasp—at least for now—we have to employ complementary approaches with synergistic effects to eradicate rim cells for good. In this article, we discussed how the combination of VTAs with different approaches, including chemotherapy, radiotherapy, PDT, bacteria therapy, immunotherapy, and RIT, led to the additive effects, and with the aid of precise drug timing, sequencing, and scheduling, CRs of tumor were accomplished in a large number of treated animals. Besides, using ligand-directed VTAs, including ITs, ICs, and coaguligands, was approved as a worthy alternative approach over combination therapies that could perform curative effects as single modalities. We believe that focusing on novel strategies that specifically and effectively enable us to target tumor rim cells will enhance the prognosis of current anti-cancer strategies and may facilitate translation of beneficial effects accomplished in the labs to the patient’s bedside.

Footnotes

Acknowledgements

The authors sincerely appreciate professor Jain for his kind permission. R.J.E. and K.S. participated in writing the manuscript and N.Z. has edited the final draft.

Consent for publication

Consent for using Figure 1 with minor changes is granted by Professor Rakesh Jain.

Declaration of conflicting interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

We wish to thank Drug Applied Research Center, Tabriz University of Medical Sciences (IR) (5/79/4301) and Iran National Science Foundation (INSF) for financial support of this study (90007316).

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