Angiogenesis in Lymph Nodes Is a Critical Regulator of Immune Response and Lymphoma Growth

Abstract

Tumor-induced remodeling of the microenvironment in lymph nodes (LNs) includes the formation of blood vessels, which goes beyond the regulation of metabolism, and shaping a survival niche for tumor cells. In contrast to solid tumors, which primarily rely on neo-angiogenesis, hematopoietic malignancies usually grow within pre-vascularized autochthonous niches in secondary lymphatic organs or the bone marrow. The mechanisms of vascular remodeling in expanding LNs during infection-induced responses have been studied in more detail; in contrast, insights into the conditions of lymphoma growth and lodging remain enigmatic. Based on previous murine studies and clinical trials in human, we conclude that there is not a universal LN-specific angiogenic program applicable. Instead, signaling pathways that are tightly connected to autochthonous and infiltrating cell types contribute variably to LN vascular expansion. Inflammation related angiogenesis within LNs relies on dendritic cell derived pro-inflammatory cytokines stimulating vascular endothelial growth factor-A (VEGF-A) expression in fibroblastic reticular cells, which in turn triggers vessel growth. In high-grade B cell lymphoma, angiogenesis correlates with poor prognosis. Lymphoma cells immigrate and grow in LNs and provide pro-angiogenic growth factors themselves. In contrast to infectious stimuli that impact on LN vasculature, they do not trigger the typical inflammatory and hypoxia-related stroma-remodeling cascade. Blood vessels in LNs are unique in selective recruitment of lymphocytes via high endothelial venules (HEVs). The dissemination routes of neoplastic lymphocytes are usually disease stage dependent. Early seeding via the blood stream requires the expression of the homeostatic chemokine receptor CCR7 and of L-selectin, both cooperate to facilitate transmigration of tumor and also of protective tumor-reactive lymphocytes via HEV structures. In this view, the HEV route is not only relevant for lymphoma cell homing, but also for a continuous immunosurveillance. We envision that HEV functional and structural alterations during lymphomagenesis are not only key to vascular remodeling, but also impact on tumor cell accessibility when targeted by T cell–mediated immunotherapies.

Full text

Angiogenesis in Lymph Nodes Is a
Critical Regulator of Immune
Response and Lymphoma Growth
Lutz Menzel
1
*, Uta E. Höpken
2
and Armin Rehm
1
*
1
Translational Tumor Immunology, Max Delbrück Center for Molecular Medicine, Berlin, Germany,
2
Microenvironmental
Regulation in Autoimmunity and Cancer, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
Tumor-induced remodeling of the microenvironment in lymph nodes (LNs) includes the
formation of blood vessels, which goes beyond the regulation of metabolism, and shaping
a survival niche for tumor cells. In contrast to solid tumors, which primarily rely on neo-
angiogenesis, hematopoietic malignancies usually grow within pre-vascularized
autochthonous niches in secondary lymphatic organs or the bone marrow. The
mechanisms of vascular remodeling in expanding LNs during infection-induced
responses have been studied in more detail; in contrast, insights into the conditions of
lymphoma growth and lodging remain enigmatic. Based on previous murine studies and
clinical trials in human, we conclude that there is not a universal LN-specific angiogenic
program applicable. Instead, signaling pathways that are tightly connected to
autochthonous and infiltrating cell types contribute variably to LN vascular expansion.
Inflammation related angiogenesis within LNs relies on dendritic cell derived pro-
inflammatory cytokines stimulating vascular endothelial growth factor-A (VEGF-A)
expression in fibroblastic reticular cells, which in turn triggers vessel growth. In high-
grade B cell lymphoma, angiogenesis correlates with poor prognosis. Lymphoma cells
immigrate and grow in LNs and provide pro-angiogenic growth factors themselves. In
contrast to infectious stimuli that impact on LN vasculature, they do not trigger the typical
inflammatory and hypoxia-related stroma-remodeling cascade. Blood vessels in LNs are
unique in selective recruitment of lymphocytes via high endothelial venules (HEVs). The
dissemination routes of neoplastic lymphocytes are usually disease stage dependent.
Early seeding via the blood stream requires the expression of the homeostatic chemokine
receptor CCR7 and of L-selectin, both cooperate to facilitate transmigration of tumor and
also of protective tumor-reactive lymphocytes via HEV structures. In this view, the HEV
route is not only relevant for lymphoma cell homing, but also for a continuous
immunosurveillance. We envision that HEV functional and structural alterations during
lymphomagenesis are not only key to vascular remodeling, but also impact on tumor cell
accessibility when targeted by T cell–mediated immunotherapies.
Keywords: lymphoma, B cell malignancy, angiogenesis, lymph node, tumor microenvironment, reactive
endothelium, lymphocyte trafficking, high endothelial venule
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5917411
Edited by:
Martina Seiffert,
German Cancer Research Center
(DKFZ), Germany
Reviewed by:
Tanja Nicole Hartmann,
University of Freiburg Medical Center,
Germany
Patricia Perez Galan,
Institut de Recerca Biomèdica August
Pi i Sunyer (IDIBAPS), Spain
*Correspondence:
Lutz Menzel
lutz.menzel@mdc-berlin.de
Armin Rehm
arehm@mdc-berlin.de
Specialty section:
This article was submitted to
Cancer Immunity
and Immunotherapy,
a section of the journal
Frontiers in Immunology
Received: 05 August 2020
Accepted: 19 October 2020
Published: 03 December 2020
Citation:
Menzel L, Höpken UE and Rehm A
(2020) Angiogenesis in Lymph Nodes
Is a Critical Regulator of Immune
Response and Lymphoma Growth.
Front. Immunol. 11:591741.
doi: 10.3389/fimmu.2020.591741
REVIEW
published: 03 December 2020
doi: 10.3389/fimmu.2020.591741

INTRODUCTION
Lymph nodes (LNs) are strategically positioned hubs of the immune
system, connecting the lymphatic system with the blood circulation,
filtering antigens and organizing the encounter of lymphocytes with
antigen presenting cells (APCs). The LN parenchyma is tightly
packed with numerous types of immune cells and susceptible for
their immigration and release during conditions of homeostasis,
inflammation and tumor transformation. The complex reciprocal
interactions of stromal cells and immune cells in LNs shape an
adapted microenvironment that supports angiogenesis and
increased LN vascularization (1). Although numerous studies
reported vascular remodeling and expansion in LNs upon
pathogen or tumor cell encounter, the detailed mechanisms and
the participating cells of these angiogenic processes are not yet
identified. In this review, we delineate the current state of knowledge
and propose probable cellular interactions that participate in
vascular growth in LNs. In particular, we will focus on the
intricate relationship between immune cells and vascular cells as a
majorpillarofthetumormicroenvironment(TME).
B cell non-Hodgkin lymphoma (B-NHL) is a heterogenous group
of hematological malignancies that arise from B lymphocytes at
various stages of differentiation. Lymphomas grow in the bone
marrow and in the secondary lymphatic organs (SLOs), with a
predominance of LNs and spleen, but they can also manifest in
non-lymphoid tissues (2). The genetic and epigenetic alterations and
the intracellular pathway dysregulations responsible for the
pathogenesis and progression of lymphomas have been extensively
studied and led to tremendous advancements in therapeutic
intervention strategies (3). The idea of tumor dependency on cells
in the surrounding of a a priori benign environment and on adapted
organ properties goes back to Rudolph Virchow in the 19
th
century
(4). The crucial influence of the cellular context in which lymphoma
cells arise and lodge attracts growing interest, and the investigation of
the TME became an increasingly appreciated field in cancer research
(5,6). The TME constitutes about half of the tumor mass in indolent
follicular lymphoma (FL) and marginal zone lymphoma (MCL),
whereas the proportion in aggressive diffuse large B cell lymphoma
(DLBCL) is generally lower and scarce in Burkitt’slymphoma(BL)
(7). On the extreme, in classical Hodgkin lymphoma (cHL) only
about 2%–3% of the cells comprise the malignant Hodgkin-Reed-
Sternberg cells (8).Hence, the composition and the dependency of the
different B-NHL and cHL on the TME differ substantially between
the entities (7). What distinguishes solid tumors and their metastasis
most from lymphoma is that within SLOs, transformed B cells
encounter a TME infrastructure that genuinely supports survival of
benign B cells. These tissues undergo refinement to the needs of the
tumor cells induced by a continuous reciprocal crosstalk of tumor,
immune and mesenchymal stromal cells (9).
The complex interactions of transformed B cells and the TME lead
to extensive changes of the vasculature within the affected organs,
which is considered to have a substantial prognostic impact on the
patients’disease outcome (7,10). The stromal compartment, mainly
comprised of blood vessels, lymphatic sinuses and the fibroblastic
reticular network is tightly interconnected and regulated. In some
respect, it can be considered to represent a joint structural
compartment in which its distinct subcompartments grow and
remodel in a synchronized manner (11,12). While the crucial role
of lymphatic vessels during lymphoma growth and dissemination is
undisputed (13,14), here we will highlight the influence of blood
endothelial cells (BECs) and the blood vasculature, which comprise
the main provider of nutrition for proliferating and differentiating
immune and tumor cells. In addition, the blood vasculature shapes a
major dissemination route for benign immune and transformed cells
(15,16).
EXPANSION OF BLOOD VASCULATURE
IN LNs DURING DEVELOPMENT,
INFLAMMATION, AND CANCER
Tumors often recapitulate developmental traits of tissues in which
they arise. The stem cell-like phenotype of many tumors is
characterized by gene expression signatures that are associated with
embryonic stem cell identity and underlines the close transcriptional
relationship between neoplastic and developmental tissue (17,18).
Similar to rapidly developing and growing organs, tumors require
blood vessels to access oxygen and nutrients. The initiation of blood
vessel expansion, referred to as angiogenic switch, occurs at different
stages during tumorigenesis, depending on the tumor type and the
respective TME. The onset of neo-vascularization and vascular
remodeling is a multifactorial processes orchestrated by activating
and inhibiting factors whose balance determines whether BECs stay
quiescent or get activated (19).
Therefore, it is useful to recapitulate the essential steps during
development to understand the basal mechanisms of the
microenvironmental remodeling in LNs. Blood vessels in LNs
reside within the stromal scaffold and are crucial for the delivery of
oxygen, nutrients, and cells. The critical delivery function was
demonstrated by the rapid occurrence of necrosis in LNs upon
ablation of the arterial feeding vessel in rats (20). During
development (Figure 1), LNs evolve from budding lymphatic veins
that form a primordial lymph sac, also known as LN anlagen. Studies
with transgenic mice lacking lymphatic vessels due to the deficiency
for the transcription factor (TF) Prox1 or appropriate
lymphangiogenesis factors, e.g., vascular endothelial growth factor-
c(Vegfc
+/−
), revealed a compromised LN development (21,22). LN
anlagen recruit hematopoietic lymphoid tissue-inducer (LTi) cells,
which in turn stimulate local mesenchymal cell differentiation into
lymphoid tissue-organizer (LTo) cells. The accumulation and
interaction of lymphotoxin (LT) a
1
b
2
on LTi cells and LTb
receptor (LTbR) expressing LTo cells results in a self-amplifying
loop of LTi recruitment and LTo differentiation that drives the LN
development (23). The lymphoid organogenesis is accompanied by
the maturation of blood vasculature driven by locally generated
retinoic acid (RA) (24). RA is presumably provided by neurons
localized adjacent to the developing LN. It directly regulates the
proliferation of endothelial cells, but also induces CXCL13 expression
in LTo cells via binding to the RA receptor-related orphan receptor
(RORgt). CXCL13 in cooperation with its receptor CXCR5 is the
exclusive inducer of the initial clustering of LTi cells in LN anlagen
independently of LT-LTbR signaling (25). A ubiquitous expression of
the mucosal addressin cell adhesion molecule-1 (MadCAM-1) on
Menzel et al. Angiogenesis in Lymphoma
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5917412

developing venous blood vessels in the LN mediates the directed
immigration of the a4b7 integrin expressing LTi cells (26–28).
Notably, the expression of MadCAM-1 in peripheral LNs of
newborns switches during the formation and maturation of high
endothelial venules (HEVs) into the expression of the peripheral
node addressin (PNAd). PNAd expression marks the completion of
the maturation of the postcappillary vessels to highly differentiated
HEVs that provide all prerequisites for the functional transmigration
of blood-borne lymphocytes into the developing and homeostatic LN
(Figure 1)(26).
The vascular system of LNs in adult mammals is composed of
arteries, capillaries, post-capillary venules and veins (29).
Arteries are characteristically located at the periphery of the
LN. The feeding arteriole enters the LN at the hilum and exhibits
a gradual decrease in diameter at its few branching points
alongside the medullary cords until it reaches the subcapsular
capillary network. Capillaries form a dense network under the
subcapsular sinus and around the medullary cords, whereas they
are markedly less dense in cortex regions and sparse within the
paracortex under homeostatic conditions. The vessel diameter
abruptly increases at the capillary to post-capillary transitions.
These post-capillary venules, referred to as HEVs, are primarily
located within the cortex in the interfollicular space. HEVs form
loop-like structures following a centripetal course that ends in
FIGURE 1 | Lymph node vascularization in development and under homeostatic conditions. The LN compartments during LN development (left) and homeostatic
conditions (right). Left: Lymphoid organogenesis is driven by recruitment of Lymphoid tissue-inducer (LTi) cells that stimulate lymphoid-organizer (LTo) cells via
lymphotoxin (LT) a
1
b
2
-LTbreceptor signaling, which secrete LTi-recruiting CXCL13 in turn. LTi recruitment from the blood circulation and the afferent lymphatics
accumulates LTi cells within the LN anlagen resulting in a self-amplifying process of LN development. a
4
b
7
integrin-expressing LTi recruitment and extravasation
utilizes the mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) on the luminal surface of blood vessels. MadCAM-1 switches to peripheral node
addressin (PNAd) expression during differentiation of mature high endothelial venules (HEVs) within peripheral LNs. The formation of the blood vessel network
comprises sprouting and branching of expanding blood vessels driven by retinoic acid (RA) stimulation of the RA receptor (RAR) on blood endothelial cells (BECs).
Right: The blood circulation enters the LN during homeostatic conditions via the feeding arteriole at the LN hilum, proceeds along the medullary cord and branches
into metarterioles that feed the capillary networks around the medulla and at the subcapsular sinus. HEVs are post-capillary venules with a characteristically enlarged
vessel diameter. The venous backflow leaves the LN in a bundle of venules at the hilum. Bottom: Representative histochemistry sections (vessels:
Cadherin5
fluoresent_reporter
, red) of murine LNs during homeostasis and during progression of a murine high-grade B cell lymphoma.
Menzel et al. Angiogenesis in Lymphoma
Frontiers in Immunology | www.frontiersin.org December 2020 | Volume 11 | Article 5917413

transitions to veins at the corticomedullary junctions. Finally, a
bundle of larger main veins leave the LN though the hilum
(Figure 1)(29,30).
Tumor growth is often accompanied by the ingrowth of blood
vessels and the formation of a vascular network, consistent with the
need for malignant cells to have access to the circulation system.
Tumor vascularization occurs either through co-option of the pre-
existing vasculature, or by induction of neovascularization. Vessel
co-option is a non-angiogenic process in which tumor cells utilize
pre-existing blood vessels of surrounding tissue to support tumor
growth, survival and metastasis (31). In contrast,
neovascularization involves a series of complex and sequential
events: (I) activation of microvascular endothelial cells, (II)
enzymatic degradation of the vascular basal membrane, (III)
gradual degradation of other extracellular matrix (ECM)
components, (IV) endothelial cell migration and proliferation,
(V) lumen formation within neo-sprouts, (VI) branching of the
neo-vessel, and (VII) formation of a functional vessel network by
fusion with neighboring vessels to initiate blood flow (32,33).
Located at the leading edge of the vascular sprout, tip cells form
cellular protrusions or filopodia to guide migration toward a source
of angiogenic growth factors. Simultaneously, they signal to
adjacent endothelial cells via Delta-like ligand (DLL)-Notch
interactions not to adapt the tip cell phenotype, but to maintain
the proliferative stalk cell phenotype and toform a vascular lumen
(34,35). The vascular endothelial growth factors (VEGFs) are the
major contributors to angiogenesis. The local secretion of VEGF-A
andits gradientformingdepositionon theECMtriggersendothelial
tip cell formation via binding to VEGFR2, resulting in endothelial
cell proliferation and migration and eventually, formation of tube
structures resembling new capillaries (35–38). VEGF-B, VEGF-C,
and VEGF-D are other members of the VEGF family of which
VEGF-C plays a critical role upon LN remodeling because it is the
most potent inducer of lymphangiogenesis as a ligand of VEGFR3.
VEGFR3 is known for its involvement in physiological and tumor-
associated lymphangiogenesis and lymphatic metastasis (39,40).
Apart from lymphatics, VEGFR3 is highly expressed at the leading-
edge of BECs that undergo sprouting (41) and was recently shown
to coregulate the expansion of the blood vessel network in LNs in a
Myc-driven high-grade B cell lymphoma mouse model (42).
Fibroblast growth factors (FGFs) stimulate endothelial cell
migration and proliferation in a very potent manner, which in in
vitro experiments evenexceeds the stimulation capacity of VEGF-A
(43,44). FGF-1 stimulates proliferation anddifferentiation of all cell
types necessary for the formation of arterial vessels, including
endothelial and smooth muscle cells. The angiogenic potency of
FGFs extends to prompt fibroblastic cells (e.g., pericytes, smooth
muscle cells, and mural cells) and recruits them for vesselformation
and maturation during tumorigenesis(45). FGF-2, the second most
abundant growth factor of the FGF family, promotes endothelial
cell proliferation and the physical organization of the endothelial
cell tube-like formation during developmental vessel assembly
(46,47).
The integral investigation of the highly complex vascular
network and the unique features of its parts in context of the
compartmentalized architecture of theLN has long been a challenge
for microscopic image analysis. Because higher order anatomical
data sets were obtained from such advanced optical imaging
approaches, algorithms for data handling were also demanding to
generate. Over the last couple of years, novel tissue preparation
methods (48,49), imaging systems and computational rendering
strategies evolved, which enable contextual and organ-wide
topological analyses in three-dimensional spaces and over time.
In particular, optical projection tomography (OPT) and light sheet
microscopy have been established to study anatomical and
functional features of LN, e.g., to quantify capillary and HEV
structures and their contextual relationship to B cell follicles and
dendriticcells (DCs) throughout the organ (50–52). A combination
of microscopic imaging and computational modulation of the
hydrodynamic properties of vessels in LNs revealed a tight
connection of the hydraulic conductivity between lymphatic and
blood vessels and the respective hydrodynamic conditions within
the LN. These biophysical conditions are vital for inter- and intra-
LN transport mechanisms and immunological functions, and most
likely for lymphoma B cell dissemination and immunosurveillance
as well (53,54). Up to date, these dynamic conditions are not easy to
mimic in organoid models. However, in an early 3D organoid
model mimicking a LN exposed to tissue injury or inflammation,
the interstitial flow affected the fibroblastic reticular cells (FRCs)
that enwrap conduits transporting fluid from the subcapsular sinus
to HEVs. Blocking this flow led to CCL21 downregulation,
indicating that increased lymph flow as a hydrodynamic factor
acts on the paracortex and thus, affects the remodeling and
functionality of conduits and FRCs (55). In line, mechanosensing
of conduit flow deprivation by FRCs in Peyer’s patches resulted in
dysfunctional HEVs and disturbed mucosal immune responses
(56). Similar processes are also conceivable during lymphoma
growth within LNs, where a gradual loss of HEVs in numerous B-
NHL was described many years ago (57). A comprehensive and
continuous blood vessel network of LNs under homeostatic
conditions has been revealed (54,58) and brought up an analysis
pipeline for detailed and whole-organ investigations of the LN
vasculature upon perturbations through inflammation, lymphoma
homing and LN solid tumor metastasis. Recently, utilization of
single cell transcriptome analysis methods revealed a broad
overview of the heterogeneity of ECs throughout several different
murine organs, including the spleen and LN as representatives for
SLOs (59,60).
THE BLOOD VASCULATURE
IS PART OF THE REACTIVE
STROMAL INFRASTRUCTURE
DURING INFLAMMATION AND
CANCER DEVELOPMENT
Inflammation, vessel reorganization and angiogenesis are
intimately connected processes. In adults, angiogenesis usually
occurs during pathological settings such as infection, wound
Menzel et al. Angiogenesis in Lymphoma
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healing and cancer. Notably, hematopoietic cells and endothelial
precursors share common CD34
+
stem and progenitor cells (61).
Growthof solidtumorsis typicallyassociatedwithinflammation
that triggers tissue-protective and pro-tumorigenic mechanisms.
Inflammatory responses in normal tissue and cancer are initiated
and maintained by local tissue or cancer associated macrophages
(TAMs) and DCs. Sustained inflammation further leads to
recruitment of bone marrow–derived monocytes, neutrophilic
granulocytes, myeloid-derived suppressor cells (MDSC), and
tissue or tumor infiltration of lymphocytes from the SLOs.
Especially cytokines and chemokines, transcriptionally regulated
downstream of NF-kB signaling pathways in immune cells,
promote cell survival and proliferation, recruit more immune
cells and re-shape the TME. Pro-inflammatory cytokines like IL-
6, TNFaand IL-17, increase the proliferation rate of other
inflammatory immune cells and prime the tumor to overcome
suboptimal microenvironmental conditions including lack of
nutrients, growth factors and hypoxia (62). Inflamed tissue and
solid tumors are often characterized by insufficient oxygen supply
that triggers angiogenesis. Hypoxia, which is the major driver of
vascular alterations in solid tumors, stabilizes the TF HIF-1a,the
master regulator of pro-angiogenic factor expression such as
VEGFs, CXCL12, and COX-2 (63–65). The presence of a
constant pro-angiogenic milieu in solid tumors often causes a
disturbed maturation and pruning of blood vessels. The division
inarterioles,capillariesandvenulescanbe deficientandresultsin an
aberrant distribution of vessel caliber, influencing the blood flow.
Morphologically, a poorly organized, malformed vessel network
develops under these conditions (66,67). The endothelial junctions
in such malformed networks are often defective and lead to
enhanced permeability and elevated interstitial fluid pressure
(68). Pericytes can be partially detached and newly build blood
vessels often fail to recruit sufficient pericyte coverage, causing an
unevenly distributed basement membrane, vessel fragility, and risk
of hemorrhage (69,70). Besides the structural and functional
defects, the specific transcriptional response of tumor vasculature
is not only related to angiogenesis and vessel integrity, but affects
endothelial activation and recruitment of leukocytes as well. Pro-
angiogenic signaling leads to endothelial anergy, reduced response
to pro-inflammatory signaling and decreased expression of
adhesion molecules and chemokines necessary for capture and
trans-endothelial migration of leukocytes (71,72).
In LNs, which are the authochthonous environment for most
B-NHL, the pre-existing vasculature takes part in the massive
remodeling process during immune responses, best studied for
strong inflammatory stimuli in mice (50,73,74). LNs are plastic
organs able to expand to a multiple of their normal size within
days including an extensive remodeling of the vascular-stromal
compartment. The rapid expansion of the LN size and cellularity
includes early events of remodeling of the feeding artery, causing
an increased blood flow and LN hypertension accompanied by
an increase of the vascular permeability (75,76) and increased
interstitial pressure. The capillary network within the cortex and
medulla expands toward the paracortex, and post-capillary
venules are reorganized (30). Skin allograft-draining LNs in
rats exhibited a progressive elongation and branching of HEVs
resulting from focal proliferation of endothelial cells in the
transition zone from high to low endothelium (77). Several
years later, Bajenoff and colleagues revisited these observations
and investigated the BEC proliferation applying a multicolor
fluorescence fate-mapping mouse model. They found similar
proliferation foci in post-capillary venules as proposed by
Anderson and Anderson. In addition, an extensive expansion
of the LN vasculature relying on the sequential assembly of
endothelial cell proliferative units upon inflammation was
observed. Clonally proliferating HEV cells (73) and capillary
resident precursors (60) comprised local progenitors for HEV
elongation and capillary neo-vessels during BEC turnover and
vessel sprouting. Interestingly, recruitment of bone marrow–
derived endothelial cell progenitors did not contribute to the
local LN vascular alterations in this model. LN expansion
stimulated by several immunization strategies in mouse
experiments, e.g., bone marrow–derived DCs (78), ovalbumin/
complete Freund’s adjuvant (OVA/CFA) (79), OVA/alum (80),
oxazalone (11), and lymphocytic choriomeningitis virus
(LCMV) infection (50) indicated similar courses of vessel
expansion, starting with early proliferation events that last for
up to 5–8 days. The remodeling eventually ends with a gradual
re-establishment of the vascular endothelial cell quiescence, a
normalization of the vascular bed and restoration of the normal
LN size (30,73,78).
IMMUNE CELLS ARE MEDIATORS
OF ANGIOGENESIS
Both innate and adaptive immune cells have an intricate
relationship with angiogenesis. They are involved in regulation of
BECproliferation,migrationand activationand theyprovidea large
spectrum of pro-angiogenic mediators apart from their genuine
immunological function. Hence, immune cells induce, support or
antagonize angiogenic processes during inflammation and tumor
growth (Figure 2 and Table 1)(124,125). Angiogenesis is also
importantfor the progression of B cell lymphoma, howeverthe role
of angiogenic factors and the composition of pro-angiogenic
immune cells within LNs varies between different entities.
A leading immune cell source for growth factors and chemokines
to promote angiogenesis under inflammatory and tumorous
conditions are myeloid cells (126). Macrophages are phagocytic
immune cells and important regulators of tissue homeostasis,
morphogenesis and repair. In LNs, macrophages are an abundant
immune cell population that is divided into subcapsular sinus
macrophages (SSM), medullary sinus macrophages (MSMs), and
medullary cord macrophages (MCMs) (127). Monocytes from the
blood stream and macrophages from LN remote tissues (e.g., bone
marrow)infiltrate the LN attracted by a variety of chemotactic factors,
among others CCL2, CXCL12, and the macrophage migration
inhibitory factor (MIF) (128–130). Tumor-associated macrophages
(TAMs) play a prominent role during progression of chronic
lymphocytic leukemia (CLL) by supporting tumor cell survival
(131) and regulation of the TME (132). The presence and
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polarization of macrophages during CLL is critical for the tumor
progression, as indicated by a CLL-associated skewing of T cells
toward antigen-experienced phenotypes and T cell exhaustion, which
could be reversed by monocyte and macrophage depletion. Thus,
interference with macrophage polarization in CLL turned out to be a
promising target for immunotherapy (133). Similar to TAMs in
leukemia, macrophages likely support angiogenesis in lymphoma as
well, both in cHL and B-NHL. M2-polarized macrophages induce an
immunosuppressive milieu in cHL, comprising the majority of the
PD-L1 expressing cells, located in close proximity to the Hodgkin
Reed Sternberg (HRS) cells (134). In this disease-defining tumor cell
population, high frequencies of alterations on chromosome 9p24.1,
involving copy number gain and amplifications, have been shown to
increase the abundance of the PD-1 ligands, PD-L1 and PD-L2 (135).
Furthermore, Epstein-Barr virus (EBV) infection can increase
expression of PD-1 ligands in cHL as well (136). The TAM derived
PD-L1 in conjunction with the HRS-cell derived PD-1 ligands PD-L1
and PD-L2 may neutralize the anticancer activity of PD-1+ T cells
and natural killer cells, a process that can be reversed by utilizing PD-1
blocking antibodies (137). TAMs were also frequently found in FL
and DLBCL, among them often polarized and pro-angiogenic M2-
like macrophages, which secrete angiogenic factors and re-arrange
the ECM by matrix metalloproteinase (MMP) release for vascular
expansion (138,139).
FIGURE 2 | Lymphoma induced angiogenesis in LNs and participating immune cells. Top: The LN compartments represented under homeostatic conditions (left)
and lymphoma-activated angiogenesis (right). Lymphoma growth is characterized by a strong LN volume expansion and blood vasculature growth. Remodeling of
the stromal infrastructure involves an increase of the microvessel density (MVD), as effectuated by direct angiogenic stimulation through lymphoma B cells cells, but
concomitantly also through reciprocal crosstalk of cells in the TME and recruited immune cells. Notably, the initiation of the angiogenic switch in lymphoma is
independent from hypoxia-induced HIF1apathway activation. Tumor polarized DCs (CEBP/b
high
) control the HEV differentiation status via LTa
1
b
2
and LIGHT
presentation; they release IL-1band hereby take part in the blood vessel growth by inducing VEGF-A expression in FRCs. They also secrete the angiogenic factors
VEGF-A and FGF2. B cells express LTa
1
b
2
, which exerts minor effects on HEVs, but a predominating stimulatory effect on FRCs. Expression of the chemokines
CCL2, CXCL12, and MIF recruits additional immune cells into the LN. Regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), M2-polarized
macrophages, neutrophils and mast cells are capable of producing the pro-angiogenic factors VEGF-A, VEGF-B, VEGF-C, MMP9, IL-8, IL-10, TGFb, and FGF1/2.
Bottom left: HEVs express PNAd, CCL21, and ICAM1 and thereby constitute the transmigration routes for lymphocytes under homeostatic conditions. Interaction of
CD62L, CCR7, and LFA-1 on naïve lymphocytes with these HEV-associated surface receptors and chemokines initiates lymphocyte rolling, HEV wall adhesion and
eventually, transmigration into the LN parenchyma. Bottom middle: Inflammatory vessels in reactive LNs recruit activated lymphocytes by CXCL9 secretion and
replace the homeostatic receptors on endothelial cells with CD62P, CD63E, and VCAM1 that are interaction partners of leukocyte-expressed CD44, PSGL1, and
VLA4. Bottom right: The lymphoma induced expansion of the blood vessel network favors the assembly of smaller anergic endothelium that is insufficiently equipped
for lymphocyte extravasation.
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Regarding angiogenesis, macrophages play a crucial role at each
step of the angiogenic cascade, starting from blood vessel sprouting
to vessel maturation and remodeling of the vascular network. Pro-
inflammatory conditions polarize classical activated macrophages
(M1), whereas anti-inflammatory conditions give rise to
alternatively activated macrophages (M2) including angiogenesis
associated macrophages (140–142). For example, in cHL HRS-cell
derived TGF-b, IL-13 and M-CSF educate monocytes or TAMs to
become immunosuppressive M2-polarized TAMs (Aldinucci D,
Casagrande N, 2016, Cancer Letters; Skinnidder and Tak Mak,
2002, Blood).
Macrophage-derived MMP2 and MMP9 proteases cleave the
ECM to break open matrix remodeling to pave the way for
endothelial sprout migration (85). Pro-angiogenic growth factors,
such as VEGF-A, MMPs, IL-1b, FGF2, and transforming growth
factor beta 1 (TGFb1), are part of the repertoire secreted by
macrophages in a pro-angiogenic milieu (85–89). Upon hypoxic
conditions that lead to HIF1aactivation, macrophages are able to
establish capillary-like networks in which they line a vessel micro-
tunnel and express lineage aberrant endothelial markers such as
CD31, von Willebrand factor and Cadherin-5, leading to the
assumption that macrophages may transdifferentiate into ECs
under specific conditions (143,144). Macrophages also function
as cellular chaperones during anastomosis of vascular sprouts by
guiding endothelial tip cells to undergo sprout fusion (82). Such
macrophages expressed the markers tyrosine kinase with
immunoglobulin-like and EGF-like domains (TIE2) and
neuropilin-1 (Nrp1), indicating that they are M2 polarized cells
TABLE 1 | Immune cells derived pro-angiogenic factors.
Cells Condition Angiogenic Factors Reference
MFInflammation (mouse, LPS, LTA/MDP) VEGF-A/C/D (81)
Development (zebra fish) VEGF-A (82)
Inflammation (mouse, OVA/CFA) IL-1bvia FRCs (83)
Hypoxia (in vitro) VEGF, bFGF, CXCL8, COX2, HGF, MMP12 (84)
Mouse/chicken angiogenesis model MMP2, MMP9 (85)
Human Monocytes (in vitro) VEGF-A (86)
Mouse Matrigel Assay (in vivo) IL-1b(87)
Mouse Matrigel Assay (in vivo) FGF2, PlGF (88)
Human atherosclerotic plaques VEGF-A (89)
Mouse solid tumors PDGFb(90)
Squamous carcinoma VEGF-C (91)
human cell lines in vitro TP (92)
Ovarian carcinoma MMP9 (93)
Breast carcinoma VEGF-A (94)
DC OVA/CFA inflammation (mouse) VEGF-A (95,96)
Inflammation (mouse, LPS) PGE
2
(97)
LPS, PGE
2
in vitro (mouse) FGF2 (95)
development and homeostasis (mouse) LTa
1
b
2
(98,99,100)
Inflammation (mouse, OVA/CFA) IL-1bvia FRCs (83)
co-culture with NK cells (in vitro) VEGF-C (101)
Il-10 stimulation (in vitro) Osteopontin (102)
NFMouse Matrigel Assay (in vivo) VEGF-A, MMP9 (103)
Human cells, angiogenesis assay (in vitro) VEGF-A, IL-8 (104)
Mouse wound healing assay VEGF-A (105)
MC human skin VEGF-A, IL-8, MCP-1 (106)
(107)
Human lung mast cells (in vitro) MMP9, VEGF-A/B/C/D (108)
Thyroid cancer IL-8 (109)
MDSC Mouse tumor models VEGF-A, G-CSF, MMP9 (110)
Mouse melanoma model VEGF-A (111)
Mouse ovarian cancer model VEGF-A (112)
Multiple myeloma mouse model MMP9 (113)
Colorectal cancer mouse model MMP9 (114)
T cells Inflammation (mouse, OVA/Montanide) LTa
1
b
2
via FRCs (115)
HUVECs (in vitro) GM-CSF, IL-8 (116)
Ischemia mouse model and in vitro IL-10, amphiregulin (117,118)
Type 2 Diabetes Amphiregulin, IL-10 (119)
Systemic sclerosis IL-8, MMP9, VEGF-A
induce EPCs diff.
(120,121)
Hypoxia (in vitro, ovarian cancer) VEGF-A (118)
B cells Inflammation (mouse, LPS) VEGF-A (122)
Inflammation (mouse, OVA/Montanide) LTa
1
b
2
via FRCs (115)
Virus infection (mouse, LCMV) LTa
1
b
2
(50)
In vitro tube formation VEGF-A, FGF2, PDGFA (123)
MF(macrophages), DC (dendritic cells), NF(neutrophils), MC (mast cells), MDSC (myeloid-derived suppressor cells).
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with properties similar to TAMs (82,145). New blood vessels need
to undergo maturation to become functionally stable. A crucial step
in this process is the integration of new blood vessels into the
established stromal environment and the recruitment of pericytes to
strengthen vascular junctions. Macrophages are highly abundant
around new blood vessels and help to recruit pericytes by secretion
of the platelet-derived growth factor b(PDGFb)(90,146).
Dendritic cells (DCs) are sentinel cells that connect the
innate and adaptive branches of the immune system wherein
they have important roles in host defense against pathogens and
in generating anti-tumor immune responses. The classical DC
compartment of the spleen is comprised of lymphoid tissue-
resident DCs, whereas LNs also include non-lymphoid tissue-
migratory DCs (147). Especially CD11c
medium
MHCII
high
DCs are
associated with the initiation of vascular expansion after bone marrow–
derived DC (BMDC) transfer, whereas CD11c
high
MHCII
medium
DCs
accumulate later in the process and promote the re-establishment of
vascular quiescence (78). Apart from their predominant immunologic role
as professional APC population, DCs carry a wide range of angiogenic
mediatorstomodulatevascularization.Theydosobyengagingcognate
signaling receptors, such as VEGFR2 on endothelial cells or by recruiting
and stimulating adjacent cells or cells of the TME (96). LN-resident DCs
are closely associated with FRCs and sample conduit-conveyed antigens
within the paracortical and interfollicular zone, where they are located in
the proximity of HEVs (148). This spatial proximity suggests that DCs are
likely to be a link between immune cells, vasculature and mesenchymal
stromal cells. The development, maturation and lineage commitment of
DC subsets is differentially regulated by acomplex TF network, depending
on homeostatic, inflammatory, and tumorous conditions. We recently
demonstrated that the TF C/EBPbplays a crucial role in murine DC
maturation and immunogenic functionality under homeostatic and
lymphoma-transformed conditions (149). In the presence of lymphoma
cells, enhanced expression of C/EBPbin DCs was observed which
transformed them into an immature, tolerogenic and pro-tumorigenic
subtype (150). Such aberrant maturation stages may potentially affect the
angiogenic capacities of the DCs as well. The crucial role of DCs for the LN
vasculature was elaborated in several studies (12,78,79,96)andrevealed
the DC-coordinated remodeling mechanisms of blood- and lymph-
vasculature, and the FRC network as well. The DC associated increase
of VEGF-A in reactive LNs further includes stimulation of a pro-
angiogenic program in FRCs and the recruitment of blood-borne cells
that participate in the angiogenic process. IL-1bexpression by recruited
CCR7
+
CD11c
+
DCs is associated with the enrichment of VEGF-A
expressing FRCs at the border of the LN paracortex (83). The angiogenic
role of DCs in lymphoma LNs has not been investigated yet; however, in
reactive LNs, resident classic DCs produce biologically active VEGF-A
downstream of the inflammation-associated TFs HIF-1a, STAT3, and
CREB. HIF-1aand STAT3 are generally related to hypoxic conditions,
whereas CREB phosphorylation is the consequence of autocrine and
paracrine prostaglandin E2 (PGE
2
) signaling (151). The PGE
2
production
is directly connected to pathogen induced toll like receptor-4 (TLR4)
signaling and therefore, delineates the connection of infection induced LN
reactivity with angiogenic vessel formation (152). DCs also release other
classical angiogenic growth factors like FGF2, endothelin-1 (ET-1),
CXCL12, and COX-2. FGF2 activates endothelial cells and induces
VEGF-A expression in mesenchymal cells, but also recruits and activates
macrophages and mast cells that in turn exhibit angiogenic
properties (152,153). DCs further have the capacity to modulate
angiogenesis in an indirect manner through secretion of the
monocyte and granulocyte attracting chemokines CXCL8,
CXCL1, CXCL2, CXCL3, and CXCL5 (154). The recruited
myeloid cells can be triggered to secrete the pro-angiogenic IL-1b
by a signaling pathway that includes classical DC-derived
osteopontin (155). DCs are not only associated with vascular
expansion, but also with the re-establishment of vascular
quiescence and stability in the process of reinstallation of the LN
homeostasis (78).
Neutrophilic granulocytes are the most abundant type of
leukocytes throughout the body, representing the pioneering cells
that are recruited to injuries and thus, they are frontline defenders
against pathogens.Neutrophilsinfiltrate LNs guided by
inflammatory cytokines like IL-1band TNFa,thecomplement
factors C3a and C5a, along the CXCR4-CXCL12 axis, and
eventually they are also attracted by a plethora of inflammatory
chemokines (156,157) (Capucetti, Albano, Bonecchi, Frontiers in
Immunology, 2020). Neutrophils are a source ofsoluble mediators
that exert important angiogenic functions. VEGF-A, IL-8,
hepatocyte growth factor (HGF), granulocyte colony-stimulating
factor (G-CSF), and MMP9 are the most important activators of
angiogenesis produced by these cells (103,158). Interestingly,
neutrophils are able to release VEGF-A-enriched granules upon
TNFastimulation and thus, promote vessel growth during
inflammation (105). This can become an self-amplifying process
since neutrophil-derived VEGFstimulates neutrophil migration via
an autocrine amplification mechanism, a process that likely
contributes to pathological angiogenesis during inflammation and
cancer (159). Human polymorphonuclear granulocytes have been
demonstrated to directly induce the sprouting of capillary-like
structures in an in vitro angiogenesis assay, mediated by secretion
of both pre-formed VEGF from cell stores and de novo synthesized
IL-8 (104). In the murineEm-Tcl1 model, mimicking CLL, a tumor-
associated neutrophil (TAN) population with a B cell helper-like
polarization was identified. Selective depletion of these TANs
retarded leukemia progression in SLOs substantially (160).
Mast cells (MC) are hematopoietic tissue resident immune
cells that are classically recognized as the main effector cell type
of IgE-mediated immediate allergic reactions, however they are
also frequently associated with tumorigenesis (161–163).
According to their protease expression, mast cells are divided
in two phenotypical populations: the trypase
+
chymase
−
(MC
T
)
and the trypase
+
and chymase
+
(MC
TC
) cells (164,165). MC
produce several proangiogenic factors, among them VEGF-A,
VEGF-B, MMP9, and FGF-2. In addition, mast cells
chemotactically respond to VEGF-A and FGF2, indicating that
a connection between mast cell accumulation at tumor sites,
angiogenesis and tumor growth exists (166,167).
Myeloid derived suppressor cells (MDSCs) contribute to the
induction of an immune suppressive and tumor permissive
microenvironment. They are frequently found in SLOs like
spleen, but they are rare in LNs (168). However, they are able
to modulate the L-selectin expression of naïve T and B cells,
preventing efficient HEV adhesion, transmigration, and
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subsequent antigen encounter within LN parenchyma (169,170).
MDSCs promote the formation of T regulatory cells (Tregs), the
secretion of immunosuppressive IL-10 and TGF-b, and inhibit
the activity of cytotoxic CD8 T cells via expression of arginase-1
(Arg1) and inducible nitric oxidase (iNOS) within the TME of
several tumor entities including B cell lymphoma (171,172).
Moreover, MDSCs directly influence the tumor stroma by
inducing differentiation of cancer-associated fibroblasts (CAFs)
(173,174). Pro-angiogenic properties of MDSCs during tumor
progression have been reviewed recently (175). MDSCs and their
progenitors, immature myeloid cells are usually not present in
LNs during steady state conditions. However, inflammatory
conditions and tumor-derived factors (e.g., CXCL12, GM-CSF,
and CCL2) induce the activation and accumulation of MDSC in
SLOs (176–178). MDSCs exhibit numerous immunomodulatory
properties that have considerable potential to influence
angiogenic processes in LNs, either through direct triggering of
ECs, or by stimulating leukocyte and stromal cells to establish an
angiogenic milieu (175). MDSCs are able to promote tumor
angiogenesis through releasing VEGF-A and MMP9. Mouse
models suggest that MDSCs integrate into the line of vessel-
decorating endothelial cells (179). In mouse melanoma, MDSC
contribute to A2B adenosine receptor-induced VEGF-A
production (111,180). VEGF-A in turn stimulates MDSC
recruitment from the bone marrow, creating a self-enhancing
feedback loop that promotes immunosuppression and vessel
growth (112). One of the reasons why several angiogenic
tumors occur to be insensitive to VEGF-A-targeted therapy is
the presence and recruitment of MDSCs. These cells secrete high
amounts of VEGF-A which might lead to neutralization of the
VEGF-inhibition and additionally, they establish pro-angiogenic
signaling pathways involving several other cells of the TME (110,
181). Moreover, MDSCs limit T cell adhesion and extravasation
by VEGF-A stimulated suppression of endothelial ICAM-1 and
VCAM-1 expression during tumor angiogenesis (71,182).
Lymphocytes, the major regulatory and executive cell subset
of the adaptive immune response are also able to influence
angiogenesis during inflammation and cancer, although their
specific implications are still enigmatic.
T cells comprise different subsets involved in lymphomagenesis,
including naive T cells, memory T cells, and Treg cells (183). Several
negative regulators of T cell activation act as checkpoints to fine-tune
the immune response and regulate hyperactivation. Cytotoxic T
lymphocyte antigen 4 (CTLA-4) andprogrammedcelldeath1(PD-
1) are the most potent examples of T cell immune checkpoint
molecules (ICB) (184). Cancer patients often display dysfunctional
antitumor T cell responses because of the signaling pathways
downstream of these receptors. PD-1 and CTLA-4 inhibition are
subject of extended clinical studies and led already to impressive
response rates in some tumor entities, among them melanoma, non–
small cell lung cancer and for hematopoietic tumors, (184–186), in
cHL as well (187,188). By targeting abnormal formation of tumor
vessels, anti-angiogenic agents potentially result in an enhanced
infiltration of anti-tumor effector cells, making the combination of
immune checkpoint inhibitors and anti-angiogenic agents a
promising and complementary approach in cancer adoptive T cell
therapy (189). On the other hand, as a result of IFNgand IL-12
stimulation, microvascular endothelial cells express checkpoint
molecules like PD-L1 (190,191). In line, arterial vessels express
PD-L1 and PD-L2 after toll like receptor (TLR)-3 activation upon
bacterial infection (192). The regulatory and angiogenic effects of
CD4
+
T helper cells (Th cells) are strictly associated with their
differentiation. Cytotoxic CD8
+
TcellsandCD4
+
Th1 cells produce
IFNgthat restrains endothelial cell proliferation and induces
expression of the angiostatic chemokines CXCL9/10/11 in TAMs
(126,193). In vitro studies revealed that Th2- and Th17 cell-
conditioned medium triggered endothelial sprouting, whereas
medium of Th1 cultures induced vascular regression. Conditioned
medium from Tregs had a minor or no effect (116). In vivo, CD4
+
T
cells display opposing effects on vascularization depending on their
subset differentiation. Th1 cell-derived IFNgimpairs angiogenesis in
ischemic tissue, an effect that is counteracted by regulatory CD4
+
T
cells (Tregs) that antagonize the immunologic Th1 cell response by
secreting anti-inflammatory IL-10 and TGFb. Thus, Tregs display
rather indirect pro-angiogenic properties, most likely by paracrine
effects on other potentially pro-angiogenic immune cells (e.g.,
macrophages, DCs, mast cells) (119,194). T cell recruitment,
survival and functionality are highly dependent on tumor-
polarized myeloid cells and tumor-derived factors. The typical
immuno-suppressive milieu of the TME is characterized by
polarizing factors, shifting CD4+ T cell differentiation toward
CD4
+
CD25
+
FOXP3
+
Tregs. In the aggressive Myc-driven murine
lymphoma model, this polarization process is promoted by DCs
expressing increased amounts of the TF C/EBPb(144).
In ovarian cancer, Tregs were selectively recruited into the
tumor tissue via CCL22 and CCL28 production by the tumor
cells and subsequently, Treg-induced secretion of high amounts
of VEGF-A to promote endothelial cell proliferation (118,195).
A striking example for Treg recruitment represents cHL; here,
Tregs are attracted via the Hodgkin-Reed-Sternberg cell-secreted
chemokines CCL17 and CCL22, which engage the Treg-
expressed chemokine receptor CCR4 (196), or by the
chemokine CCL20 that binds to CCR6 (Baumforth,
Birgersdotter, Machado, Am J Pathol, 2008). Th cells and
cytotoxic T cells are required to mediate the anti-angiogenic
effect of IL-12. IL-12–activated lymphocytes effectuate inhibition
of tumor growth and function as anti-vascular agents that release
higher amounts of IFNgwhile they down-regulate VEGF in
neighboring cells (197,198). Noteworthy, the presence of IFNg
comes at the expense of an induction of PD-L1 on numerous
stromal cell types, among them endothelial cells (199,200); this
process is likely to counteract the beneficial effects of IFNg-
secreting effector T cells which may be rendered dysfunctional
(201). The infiltration of tumor sites by cytotoxic CD8
+
T cells is
usually correlated with a favorable clinical prognosis, however
immunosuppressive conditions can polarize these cells to
CD8
+
FOXP3
+
regulatory cells with similar immunomodulatory
and angiogenic properties as CD4
+
Tregs (202–205). Studies of
coronary artery disease and systemic sclerosis found T cells with
angiogenic potential in blood samples of patients and
demonstrated that these CD3
+
CD31
+
CXCR4
+
cells (referred to
as angiogenic T cells) play a vital role for the colony formation
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and differentiation of endothelial progenitor cells (EPCs) in the
bone marrow (110,206). Such EPCs have been detected in the
circulation and in LN samples from patients with B-NHL as well,
although their influence on lymphoma-induced vessel growth is
still elusive (207,208). However, inflammation models argue
against a significant functional role of BM-recruited EPCs in LN
vascularization (73).
B cells are frequently found to be part of the TME (209);
however, their role in tumor progression and vascularization is
still unclear. They can directly promote angiogenesis by secreting
pro-angiogenic factors such as VEGF-A, FGF2, and MMP9
(210), or indirectly by polarizing macrophages to the M2 pro-
angiogenic phenotype (211). Transgenic mice (CD19
Cre
/hVEGF-
A
fl
) overexpressing human VEGF-A in murine B cells exhibited a
VEGF-A induced lymphangiogenesis and an expansion of HEVs
in LNs. The authors of the study speculated that the
unphysiologically high levels of human VEGF-A might not
directly influence the LN lymph- and blood vasculature, but
may rather cause an accumulation of pro-angiogenic
macrophages (122). In a mouse model of LCMV infection, B
cells were shown to be required for LN tissue remodeling and
vessel expansion. Surprisingly, the latter was independent of
VEGF-A signaling pathways, but required LTa
1
b
2
-expressing B
cells (50). A recent study emphasized the angiogenic capacity of a
B cell subset during eosinophilic esophagitis and in patients with
melanoma. These pro-angiogenic B cells were identified by the
surface markers IgG4
+
CD49b
+
CD73
+
and shown to promote
vascular tube formation in vitro through VEGF-A, FGF2, and
PDGFA expression (123). Taken together, although B cells
express VEGF-A and LTa
1
b
2
during certain conditions, their
role in LN angiogenesis is not well understood. Potentially, B
cells may exert pro-angiogenic effects themselves, but also
through stimulation of other stromal cell types, such as FRCs
and macrophages (Figure 2).
B CELL LYMPHOMA-INDUCED
VASCULAR CHANGES ARE DEPENDENT
ON THE ENTITY AND STATE OF
LYMPHOMA PROGRESSION
The clinical importance of angiogenic processes and mechanisms
for the growth of solid tumors is well recognized (212,213).
Therapeutic concepts from solid tumors targeting the VEGF-A/
VEGFR1/2 axis have been adopted for combinatorial therapies of
B-NHL, resulting in rather disappointing clinical outcomes (214,
215). We recently showed that angiogenic processes in LNs in a
mouse model of high-grade B cell lymphoma are induced by
signaling pathways distinct from solid tumors. In sharp contrast
to most solid tumors, lymphoma growth in LNs was not
associated with hypoxic conditions or inflammation. Instead,
lymphoma affected vessel expansion via the VEGF-C/VEGFR3
and LTa
1
b
2
/LTbR signaling axes (42). In patients, the growth of
tumor cells in low-grade B-NHLs is usually exponential for a few
months and remains in a steady state as indolently growing
tumor mass for years. This indolent lymphoma is considered to
be avascular with dormant endothelial cells within the TME. In
contrast, high-grade B-NHL progression is often accompanied
by a so called “vascular phase”, which represents extensive
vascularization of LNs (216,217). Such intermediate- and
high-grade B-NHLs grow exponentially without intermission
phase until they reach a mass critical for a patient’s survival.
As a clinical indicator of the vascularization, B-NHLs are usually
quantified by terms of the microvessel density (MVD).
Immunohistology using anti-CD31 antibody staining is still
considered the “gold standard”of blood vessel detection, even
though there is substantial variation between different studies
due to the heterogeneity of the lymphoma stroma and different
scoring methodologies. In some cases, the marker CD34 is used
to detect the blood vasculature. Notably, lymphatic vasculature
also expresses CD31, but at much lower levels (42,218,219).
Non-invasive assessment of tumor vascularization in vivo is
possible by using Doppler sonography, contrast-enhanced
dynamic magnetic resonance imaging (dMRI) and positron
emission tomography-computer tomography (PET-CT). These
techniques do not allow a direct quantification of the blood
vessel density but provide information on the functional status
of the blood vessels, e.g., vessel integrity, permeability, perfusion
and metabolism (220). Another diagnostic approach to detect
ongoing angiogenesis in vivo is the serological quantification of
growth factors. VEGF-A levels in the serum of patients with
progressive NHL were significantly elevated in comparison to
patients in complete remission (221,222). Elevated VEGF-A levels
have been found in aggressive B cell lymphoma subtypes including
MCL, DLBCL, but also in indolent lymphoma, such as CLL and
small lymphocytic lymphoma (SLL), respectively (223–225). A
variety of commonly used B-NHL cell lines secrete measurable
VEGF amounts under serum starvation conditions, whereas other
angiogenic factors like the placental growth factor (PlGF) and
FGF-2 are not expressed (226). However, the detection of
angiogenic factors in clinical serum samples gives no
information on the cellular source of these molecules and is not
a reliable indicator of angiogenesis in the compartment of interest.
Previously, a group of angiogenesis experts published consensus
guidelines for the use and interpretation of angiogenesis assays,
which involve in vivo, ex vivo explantation, and in vitro bioassays.
They explicitly highlighted critical aspects that are relevant for the
execution of angiogenesis detection and proper interpretation (227).
Mantle cell lymphoma (MCL) is an aggressive B cell
neoplasm that comprises 6% of all NHL cases (228,229). It is
susceptible to paracrine signaling from the microenvironment
and in turn shapes the microenvironment by secreting soluble
factors (230). MCL is genetically characterized by overexpression
of the CCND1gene, encoding for cyclin D1 (231). Recent studies
identified a subgroup of MCL that has a more indolent behavior
with a clinical presentation as leukemic disease, exhibiting
minimal LN distribution and a frequent splenomegaly. These
tumors also overexpress cyclin D1 but lack expression of the sex
determining region-Y-box11 (SOX11), a TF specifically
expressedinconventionalMCLandassociatedwithan
aggressive and angiogenic phenotype (232). These results have
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been confirmed in MCL patient samples by using
immunohistochemistry, demonstrating a correlation between
an increased MVD and high levels of SOX11 expression (233).
Experiments with MCL tumor xenotransplants in mice, in cell
lines, and in primary MCL samples revealed that SOX11 actively
modulates angiogenesis by up-regulation of the platelet-derived
growth factor a(PDGFa), which is a competent inducer of an
FRC-associated pro-angiogenic program (234,235). Moreover,
SOX11 overexpression promotes B cell receptor signaling
represses Bcl6 transcription and upregulates PAX5 to avoid B
cell differentiation into memory B cells or plasma cells. PAX5
supports tumor cell homing and invasion via up-regulation of
CXCR4 and the focal adhesion kinase (FAK) (236–238). The
absolute monocyte count in MCL correlated with the prognosis
and supports the hypothesis that the TME is relevant for MCL
tumor progression (239). CD68
+
and CD163
+
macrophages were
found in MCL LNs without exception. Substantial numbers of
VEGF-C expressing macrophages were found in a mouse
xenotransplantation model as well (240). Treatment with the
immunomodulator lenalidomide depleted monocytes and
VEGF-C expressing macrophages, resulted in impaired
functional lymphangiogenesis. However, a relevant impact on
lymphoma-associated blood vessel growth in MCL was not
investigated in this study. Of note, in human MCL anti-
inflammatory and pro-angiogenic CD163
+
cells (M2-like)
outnumbered the more inflammatory CD68
+
CD163
-
macrophages (233), indicating a propensity to stimulate
angiogenesis. This M2-like polarization of macrophages is
actively driven by MCL derived CSF-1 and IL-10 (241). MCL
cells exhibit increased expression of the T cell, B cell, and
monocyte recruiting chemokines CCL4 and CCL5 compared to
normal B cells (242). T cell infiltration has been considered as a
prognostic marker in MCL in which CD8
+
, and particularly
CD4
+
T cell frequencies are higher in indolent MCL and decrease
with more aggressive histological and clinical presentation (243).
In contrast, a recent study reported an expanded vascularization
of MCL associated with a high infiltration of CD4
+
and CD8
+
T
lymphocytes (233). The differences might be explained by a weak
comparability of data that were either correlated with the clinical
outcome, or with the SOX11 expression level in MCL, two
hallmarks that are not always correlated. A more detailed T
cell characterization of CD4/CD8 T cell subsets is required for a
more reliable assessment of the T cell–related influence on
angiogenesis and the clinical outcome in MCL. Interestingly,
MCL cells itself express the VEGFR-1, providing a strong
rationale to target VEGF in order to interfere with angiogenic
processes and concomitantly, with autocrine survival signals
(230,244).
Angiogenesis is likely a part of MCL progression, driven by
MCL derived PDGFa. Therapeutical interference with PDGFR-b
signaling, the receptor for PDGFa, can be achieved with receptor
tyrosine kinase (RTK) inhibitors. Some PDGFR-btargeted drugs
have been tested in clinical trials for B-NHL but failed to bring
significant benefit(217). In contrast, immunomodulating drugs
(IMiD) like thalidomide and lenalidomide have anti-angiogenic
properties and showed great potential in combination with
rituximab for the treatment of untreated or relapsed MCL
patients (245,246).
Follicular lymphoma (FL) is the second most common B-
NHL, accounting for 20% of all B-NHL cases (247). The disease
affects LNs, spleen and frequently also the bone marrow.
Neoplastic follicles in FL have a lower proliferative index in
comparison to reactive germinal centers. However, the
proliferative capacity of FL cells increases gradually with the
FL grade. FL progression requires the supporting infrastructure
of the follicular TME to maintain survival, a requirement that
gets progressively lost in the process of transformation to
aggressive DLBCL (248,249). Follicular dendritic cells (FDCs)
are one branch of this supporting infrastructure. They are of
mesenchymal origin and represent a crucial stromal cell
population supporting the germinal center reaction and
maintenance of the B cell follicle in LN and spleen (250,251).
FDC secreted B cell survival factors such as Indian hedgehog
(HH), the B cell activating factor (BAFF), and IL-15 are
potentially pro-tumorigenic (252,253). CXCR5-controlled
access to FDCs conferred survival and proliferation stimuli to
CLL B cells in the murine Em-Tcl1 model, which mimics some
aspects of indolent tumor growth (253). Similar to reactive LN
follicles, neoplastic follicles in FL preserve the organized FDC
network structure at least in early stages of the disease
progression (254). FL-FDC cross-talk induces a pro-angiogenic
expression pattern in FL cells, including secretion of VEGF-A
and VEGF-C (255). This cross-talk is crucially dependent on the
phosphoinositide-3-kinase d(PI3Kd), providing therapeutic
intervention options with PI3K specific inhibitors like
idelalisib, which is approved for the treatment of FL, CLL, and
SLL (256). The second branch of the supportive infrastructure in
follicles are the CD4
+
CXCR5
+
PD1
+
T follicular helper (Tfh)
cells, which provide vital survival signals for FL cells by secreting
IL2, IL4, IFNg, and by CD40L presentation (9,257). FL cells are
further dependent on proliferation and survival signals of the B
cell receptor (BCR) in interaction with FDCs and TAMs (258).
Elevated numbers of M2-like TAMs are found in the immediate
microenvironment of FL cells and neo-vascular sprouts within
the follicle (138). However, the prognostic value of CD163
+
TAMs remains controversial and is highly dependent on the
prior course of treatment (259). In sum, FL appeared to be less
prone to induce relevant vascular changes, whereas LNs of high-
grade B-NHLs exhibited a dense and aberrantly distributed
vasculature within the paracortical zone. In contrast to most
other B-NHL malignancies in which high levels of pro-
angiogenic factors and an increased MVD is associated with
an adverse prognosis, high level FL vascularization correlates
with a beneficial disease course (260–262). The improved
clinical outcome apparently correlated with the increased
vascularization, but was surprisingly independent of follicular
VEGF-A expression (223,263). Some studies stated a minor
vascular remodeling in FL compared to reactive LN or follicular
hyperplasia, or even vascular regression constraining the growth
of reactive and neoplastic follicles (260,264). Therefore, the
clinical significance of angiogenesis in FL remains uncertain. In
one clinical trial, addition of the anti-VEGF bevacizumab during
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rituximab treatment of relapsed FL significantly improved the
progression-free survival (265). The potential of angiogenesis
inhibition upon treatment of FL requires further evaluation in
larger clinical trials.
Collectively, according to the data currently available it seems
that angiogenesis is important for high-grade lymphoma, but has
less impact on indolent FL growth.
Diffuse large B-cell lymphoma (DLBCL) is the most
common type of lymphoid tumors worldwide accounting for
30% of all diagnosed NHL and characterized by the large size of
neoplastic B cells and usually a very aggressive clinical
presentation (266). Lenz et al. identified gene expression
profiles in LN from patients pre-treated with the combination
therapy anti-CD20 antibody, cyclophosphamide, doxorubicin,
vincristin, prednisolone (R-CHOP), dividing DLBCL in two
distinct subgroups that are predictive of the clinical outcome
(267). The “stromal-1”signature includes expression of
extracellular matrix (ECM) elements, ECM remodeling factors
(MMP2,MMP9,M1-MMP,PLAU,TIMP2) and is associated
with a favorable prognosis. The “stromal-2”signature”was
found in tumors with an increased MVD and is characterized
by markers of endothelial cells (Pecam1,Vwf,Kdr,Tek). The
latter signature is associated with a poor clinical outcome,
emphasizing the critical impact of angiogenic processes on
aggressive B-NHL progression. Several studies investigated the
clinical consequences of the “stromal-2”signature and confirmed
the correlation of a high MVD with an adverse outcome and a
shorter overall survival rate (268–271). The relationship between
MVD and DLBCL behavior was the object of many studies and
was found to be associated with poor prognostic parameters such
as splenic involvement, high mitotic rate, and capsular invasion
(268–272). Gomez-Gelvez et al. reported contradictory results,
showing that high MVD is associated with rather better
progression-free survival (PFS) and event-free survival (EFS)
(273). Several other studies also failed to draw a connection
between the MVD, tumor grade and prognostic outcome (274–
277). A DLBCL mouse xenotransplantation model demonstrated
that the inhibition of the paracrine VEGFR-2 pathway reduced
growth of an established lymphoma and correlated with
decreased tumor angiogenesis (226). DLBCL cells often
overexpress the phosphodiesterase 4B (PDE4B), which
intracellularly catalyzes the hydrolysis of cyclic-AMP (cAMP).
The cAMP-PDE4B axis modulates signaling of PI3K and AKT
and therefore acts upstream of VEGF-A expression. Experiments
with genetically or pharmacologically inhibited PDE4B resulted
in decreased VEGF-A expression in lymphoma cells and reduced
angiogenesis in the Eµ-Myc high-grade lymphoma mouse
model (278).
In a gene expression study on relapsed or refractory DLBCL,
patients with the ABC-like DLBCL subtype that had low VEGF
121
isoform expression, exhibited a significantly better overal survival
than those with high VEGF
121
gene expression levels (279).
Interestingly, VEGF
121
low transcript levels were associated to a
gene signature reflecting immune response and T cell activation.
DCs are likely a major source of VEGF-A in LNs with DLBCL.
Functionally, DCs could be involved in lymphoma TME
remodeling, but their number in DLBCL LNs is significantly
lower than in reactive LNs. Lower expression levels of the LN
homing receptors CD62L and CCR7 in DCs in LNs of DLBCL
patients were thought to result in reduced DC immigration.
However, it remains elusive if the DCs lose the receptor
expression upon arrival in the LN, or whether these cells are
recruited via alternative routes (280). In an aggressive Myc-driven
lymphoma model in mice a tumor-specific DC differentiation
occurs that promotes tumor cell survival and favors the
maturation of monocytic-derived DCs (MHCII
medium
)(149,150).
Alongside tumor repressing M1 macrophages, “alternatively”
activated M2 macrophages exhibit angiogenic capacities, they are
frequently found in DLBCL and often correlate with a poor
prognosis (281–283). Although numerous studies reported an
association between TAMs and MVD in DLBCL, others could
not find a correlation between CD68
+
macrophages and an
increased MVD (270). Such controversies can probably be best
explained by variabilities in the methodological approaches. The
macrophage marker CD68 represents M1 and M2 macrophages
and therefore, produces inaccuracies in the interpretation of studies
concerning the macrophage-MVD correlation. The addition of the
marker CD163, which rather recognizes M2 activated
macrophages, including angiogenic macrophages, provides a
more reliable view on the role of macrophages in DLBCL (284).
Elevated numbers of macrophages have been correlated with poor
prognosis in DLBCL (282). However, in therapeutic setting
macrophages are required to confer treatment effects when
patients were treated with anti-CD20 antibody (e.g., Rituximab).
Here, macrophages mediate tumor cell depletion via the
macrophage Fc-gamma receptor (FcgR) expression (215).
Another abundant immune cell population in LNs of DLBCL
patients is mast cells with a predominance of MC
T
-type (tryptase-
positive) cells. M C
TC
-type (tryptase-positive andchymase‐positive)
and CD4
+
Th
2
were shown to express IL-4 in DLBCL and therefore,
they may actively promote survival of the tumor cells (285).
Hedström and coworkers examined 154 DLBCL cases and
suggested that the infiltration of mast cells reflects the
inflammatory immune response of the endogenous anti-tumor
defense and is therefore related to a favorable outcome (286). The
gradual increase of the MVD was correlated with an increasing
number of mast cells. Although mast cells are considered to be
bystandersin tumor immunology,additionalpro-angiogeniceffects
of these cells are likely as they secrete relevant amounts of different
VEGFs, FGF-2, trypase, and granzyme B. The latter has a pro-
angiogenic effect via the enzymatic mobilization of ECM-bound
FGF-1 (287,288). The wide range of physiological conditions and
tumor entities that include mast cell-supported angiogenesis and
the respective recruitment and signaling pathways were excellently
reviewed by Ribatti et al. (289).
Apart from the direct effect on immune and tumor cells,
surprisingly, the application of the VEGF-A inhibiting antibody
bevacizumab to R-CHOP therapy increased adverse cardiac events,
yet without increasing the therapeutic efficacy in DLBCL patients
(214,215). From these studies it can be inferred that the increased
MVD in DLBCL patients may be simply a correlation with minor
importance for the disease course, or that other non-VEGF-A
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angiogenic pathwaysprevail and cause enhanced vascular assembly
instead. Ina study of Pazgal et al., VEGF-C, VEGF-D, and VEGFR3
were expressed in both lymphoma cells and endothelial cells of the
blood and lymphatic vasculature. They reported a significant
correlation of the VEGF-C expression and the presence of blood
vessels. VEGF-D expression correlated with the patient
International Prognostic Index (IPI) Score and the patients’
overall survival (14). These results may indicate that apart from
its role as primary signaling pathway for lymphatic vessels, the
VEGF-C–VEGFR3 axis also has implications on angiogenic
processes of LN blood vessels. A study in breast cancer
demonstrated that VEGFR3 is significantly upregulated in the
endothelium of new blood vessels. The results also suggested that
VEGF-C secreted by the intraductal carcinoma cells acts
predominantly as an angiogenic growth factor for blood vessels,
although other immune or stromal cells might be involved in this
paracrine signaling network as well (290). An experimental study
using a Myc-driven aggressive lymphoma mouse model, which
resembles important aspects of aggressive B-NHL, supported this
hypothesis, showing that the MVD expansion was triggered by
lymphoma-provided VEGF-C, in a synergistic activity with
LTa
1
b
2
(42).
Representing a high-grade and angiogenesis-associated
lymphoma type, multiple clinical trials with anti-angiogenic
agents for the treatment of DLBCL have been conducted. Most of
the treatment approaches using single agent angiogenesis inhibitors
failed to prove a beneficial effect. However, combinatorial treatment
strategies such as R2-CHOP (lenalidomide, R-CHOP) (291,292),
brought encouraging results. Such oberservations emphasize that
anti-angiogenesis therapies might not be effective when applied
alone, even in highly vascularized lymphoma, but are valuable
components in combination with other drugs.
Burkitt’slymphoma(BL) represents around half of all malignant
non-Hodgkin lymphoma in children and around 2% in adults (293).
The BL pathogenicity is usually associated with the infection of B cells
with the Epstein-Barr virus (EBV). EBV gene products induce BL
cell-derived soluble factors that result in inhibition of neo-
vascularization and eventually tumor necrosis and regression (294).
However, in in vivo experiments, EBV-positive cells induced massive
recruitment of leukocytes at the tumor border and the development
of granulation tissue with large numbers of blood and lymphatic
vessels (295). Surprisingly, aggressive BL displayed the highest MVD
in comparison to intermediate DLBCL and indolent B-NHL (42,262,
287). In support of this observation, BL showed increased
vascularization relative to benign lymphadenopathies and can
produce several angiogenic factors, although it is not yet known
whether this is due to Myc gene overexpression or the EBV
transformation (296–298). BL were found to be closely associated
with VEGF-producing CD68
+
VEGFR1
+
myeloid cells located
around the neo-vasculature. The newly formed blood vessels were
identified by the absence of pericyte coverage as result of the rapid
vessel growth (299,300). Genetic depletion of this subpopulation of
CD68+VEGFR1+ myeloid cells was sufficient to inhibit angiogenesis
in experimental lymphoma (301). To our knowledge, to date there are
no clinical data or published treatment strategies of BL that target
angiogenesis specifically.
Classical Hodgkin Lymphoma (cHL) is characterized by
mono-nucleated Hodgkin and multi-nucleated Reed-Sternberg
(HRS) cells, which comprises tumors with mixed cellularity,
nodular sclerosis and lymphocyte-rich or lymphocyte-depleted
subtypes. Different from other lymphoma, HRS cells are the
minority of cells within the affected LN. Most of the cells in cHL
tumors are cells of the TME, indicating a prominent role ofbenign
immune cells and the LN stroma (302). A crucial role of
angiogenesis and increased MVD have been reported for cHL
and correlate with a poor prognosis (303). Similar to observations
in highly vascularized LNs in an aggressive B-NHL mouse model
(42) and in immunohistochemically characterized B-NHL patient
specimen (277), in cHL HIF-1awas only moderately expressed
(304), suggesting that angiogenesis in cHL is not hypoxia-driven
and may utilize other angiogenic pathways instead. In childhood
cHL, HRS cells express VEGF, MMP-2 and MMP-9. However, the
expression of these factors did not correlate with the MVD and
neovascularization level (305,306). On the other hand, VEGF-D, a
ligand for VEGFR3 and usually associated with lymphangiogenesis,
is expressed in HRS cells at high abundance and correlated with
high numbers of microvessels (307). Moreover, in vitro HRS cell-
derived TGF-b,FGF-2,andVEGFsupportedHUVEC
tubulogenesis (308,309). Secretion of Ltaby HRS cells activated
endothelial cells, which enhances adhesion molecule expression
and consequently, recruitment of T cells. This mechanism amplifies
the inflammatory milieu in the cHL TME through conditioning of
the blood vasculature (310).
Commonly attributed to the occurrence of angiogenic M2
macrophages, TAMs are linked to poor outcome in HL.
Interestingly, lack of macrophages, but also high numbers of
macrophages is associated with a poorer disease-free survival and
overall survival, whereas intermediate numbers are associated
with a better outcome. This macrophage paradox suggests that a
lack of TAMs is beneficial for HL growth, while TAMs have an
inhibitory effect with increasing numbers (311). The inhibitory
effect seems to be displaced by an adverse effect of TAM-induced
angiogenesis, supposedly predominated by CD163
+
M2-like
TAMs (312). High numbers of CD163
+
TAMs correlate with
elevated VEGF-A levels and an increased MVD, indicating that
CD163 is an independent prognostic marker in cHL (313).
Interestingly, although the particular signaling pathways within
TAMs remain elusive, pre-clinical experiments with PI3K-Akt
pathway inhibition suggested a connection to macrophage M2-
polarization (314,315), which could be a promising anti-
angiogenic intervention clue by prevention of pro-angiogenic
activity of M2-like TAMs.
ANTI-ANGIOGENIC THERAPIES IN
COMBINATION WITH CHEMOTHERAPIES
Cancer therapy earlier than the 1970s was solely focused on
targeting the actual cancer cells. Judah Folkman’s discovery that
tumor growth is angiogenesis-dependent led to a profound
paradigm shift in cancer therapy (316,317). Sprouting
angiogenesis plays an essential role in tumor growth, invasion,
Menzel et al. Angiogenesis in Lymphoma
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progression, and metastasis, targeting this process is a promising
strategy to inhibit growth and spread of solid tumors. Clinical
trials and treatment strategies of anti-angiogenesis therapy in B-
NHL were recently reviewed (217). Angiogenesis inhibitors are
classified into direct and indirect agents. Direct inhibitors target
vascular ECs and include endostatin, arrestin, and tumstatin.
Indirect angiogenesis inhibitors target tumor cells or cells of the
TME to prevent the expression of pro-angiogenic factors or
block their activity (318). The anti-VEGF monoclonal antibody
Bevacizumab was the first anti-angiogenesis drug approved by
the FDA for the treatment of metastatic colon, ovarian, renal,
non-squamous cell lung cancer, and glioblastoma multiforme.
Unfortunately, clinical significance was only reached in
glioblastoma multiforme treatment (319,320), a result that
could not be confirmed in other studies (321). In contrast to
Bevacizumab, treatment with tyrosine kinase inhibitors (e.g.,
Sorafenib) that interfere with the signal transmission of
VEGFRs resulted in remarkable effects throughout several
cancer entities. Combination of tyrosine kinase inhibitors and
conventional chemotherapy have not been beneficial (322).
Conventional chemotherapy can cause direct cytotoxicity of
endothelial cells, but this effect is non-selective and only
observed upon the maximal tolerated dose (MTD). Insufficient
tumor and vascular bed destruction can effectuate a strong
hypoxic condition, which results in release of chemoattractant
CXCL12. Accordingly, MTD chemotherapy potentially increases
systemic CXCL12, which recruits bone marrow–derived EPCs.
These cells can cause recurring angiogenesis in mouse models of
solid tumors (323,324). Therefore, an anti-angiogenesis therapy
that is complementary to chemo- or immunotherapy is aimed at
restricting pro-angiogenic bystander effects of the tumor
treatment. In addition, instead of aiming for a complete
vascular eradication, the paradigm in anti-angiogenic therapies
shifted to vascular normalization (325,326).
Rituximab has become an essential part offirst-line treatment of
several B cell lymphoma entities, foremost of DLBCL. However,
ongoing research aims to improvethe therapeutic efficiency and the
reduction of the relapse rate of drug-resistant lymphoma cells.
Tumor anti-angiogenesis therapy approaches are one branch of
such research, in whichBevacizumab and Endostatin werethe most
promising representatives for lymphoma treatment (327,328).
VEGF-A has a crucial role in promoting vessel growth, but is also
considered to be an immunosuppressive factor that modulates the
migration and function of several immune cells, e.g., DCs and mast
cells. The potential pharmaceutical targeting of the VEGF/VEGFR
axis to modulate anti-tumor immunity has been reviewed
recently (329).
An important challenge of anti-angiogenic therapy in solid
tumors as well as in lymphoma is the identification of the
particular angioactive receptors throughout different tumor entities
and individual patients. The inhibition of intracellular signaling hubs
is a strategy to overcome the targeting of distinct angiogenic tyrosine-
kinase receptors. Class I PI3Ks are involved in the signal transduction
of many pro-angiogenic signals and control cell growth, survival,
motility, and metabolism (330). PI3Kdinhibition in lymphoma
potentially also interferes with tonic signaling in tumor cells, e.g.,
via the BCR signaling pathway (331), or breaks the Treg-mediated
immune tolerance (332). Interestingly, PI3K activity is essential for
macrophage M2 polarization (333) and therefore, a potential target to
hamper M2-like angiogenic macrophages. Inhibition of PI3K
signaling represents a valuable therapeutic strategy to target
different indolent B cell lymphoma entities, among them FL, CLL,
SLL, and more recently, they showed promise in T cell lymphomas as
well (334,335). The combinatorial treatment of the first generation
PI3K inhibitor idelalisib with rituximab or bendamustine revealed
favorable response rates in FL patients (334), but serious adverse
effects due to bacterial and viral infections were observed.
Additionally, immune-mediated and hematologic adverse events
occurred. Beyond that first generation PI3K inhibitor, newer PI3K
inhibitors such as copanlisib and duvelisib were introduced for
patients with relapsed and progressive FL, CLL, SLL, respectively.
These inhibitors differ in their preference for PI3K isoforms which are
expressed differentially in various tissues (336). Despite relevant side
effects of PI3K inhibitors, they have been judged clinically
manageable and thus, prompted an FDA approval for relapsed and
refractory indolent B-NHL (335,337). Published reports on anti-
angiogenic therapies in B-NHL allow the conclusion that the complex
mechanisms of angiogenesis in lymphoma are incompletely
understood and require further pre-clinical and translational
research to develop reliable and effective anti-angiogenic treatment
strategies. Moreover, new anti-angiogenic treatment regimens need
to be validated regarding an actual reduction of tumor growth, since
sole targeting of angiogenic factors often fail to cause substantial
tumor regression (Figure 3)(340).
OUTLOOK
Vascular remodeling and angiogenesis have been increasingly
recognized as crucial factors in the pathophysiology of B-NHLs.
We here present an integrated concept that includes angiogenic
processes of the LN TME beyond the proliferation and survival
of endothelial cells stimulated by the VEGF-VEGFR axis. In
human, the angiogenic properties of LN resident and recruited
immune cells are still insufficiently understood. Studies to
address such functional states are limited because tissues from
appropriate human patients are rarely available. Notably, most of
the human data available so far are observations on clinically
progressed and even terminal stage lymphoma LNs. Flow
cytometry analysis of blood samples is usually limited to a few
entities (e.g., FL, CLL, and MCL), common markers and cannot
readily be correlated with pathohistological observations due to
the lack of tissue specimen. Availability of LN tissue from
progressed disease stages is often limited to scarce material
from fine-needle biopsies. As a useful surrogate, mouse models
of reactive LNs and LNs with lymphoma growth demonstrated
that the angiogenic processes are regulated by a timely complex
interplay of immune, tumor, and stromal cells (42).
In the future, modern methods like single-cell RNA sequencing
alone or with spatial resolution, or single-cell analysis in
combination with proteomics will help to resolve the complexity
of participating cells and their heterogenous differentiation status.
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This technique requires much less input material for a high
resolution analysis at the genome, protein, or epigenome level
(341–343). Even patient-derived specimen from fine needle
biopsies seem amenable to such analysis, allowing then a kinetic
description of LN remodeling in the course of diagnosis, treatment
response, and eventually relapse. Single-cell RNA sequencing will
further enable the discrimination of different endothelial cell
subtypes and their differentiation traits (344). The compartment
of BECs is comprised of several functionally distinguishable
subpopulations that further differentiate during angiogenesis. To
date, we know little about the role, differentiation conditions and
distribution of these subpopulations in LNs. Such transcriptional
observations need to be correlated with the topology of the single
cells and cell networks within the complex LN (345). Modern
imaging methods, e.g., light sheet microscopy and intravital 2-
photon microscopy enable the complex spatial integration and the
investigation of dynamic processes in situ, which have long been
restricted to snapshot observations. Very recently, a new generation
of flow cytometry devices became available that, based on a
spectrum wide detection of fluorophores, allow a simultaneous
detection of a multi-fold higher number of cell markers. The
possibility to determine extended marker panels with small
sample sizes will not only improve basic knowledge in the pre-
clinical context, but will also provide innovative approaches for
clinical diagnostics (346,347).
Improving insight into angiogenesis is also of considerable
relevance for the emerging immunotherapies using chimeric
antigen receptor (CAR)- and T cell receptor (TCR)-transgenic
T cells and NK cells. It is reasonable to suggest that tumor blood
vessels have a leading role in granting effector T cell access to the LN
and the tumor niche formed therein. For example, solid tumors
condition an endothelial activation status that can be considered
immunologically “silent”(348). However, reactivation of such
vessel-lining endothelial layers is a prerequisite for the adhesion
and transmigration cascade of naive and therapeutic T cell
populations. We envision that this endothelial tuning is not only
applicable to solid tumors, but also to LN-localized lymphatic
neoplasm. Except for cHL, immune checkpoint blockade (ICB)
targeting PD-1 or CTLA-4 has not shown relevant benefitinotherB
cell neoplasm. Because ICB efficacy depends on the presence of a
repertoire of antigen-specific T cells, a rational sequence of
immunotherapeutic interference in B-NHL would start with a
vessel induction toward a more activated or even inflammatory
state. It seems not even necessary to overactivate local endothelial
cells, as shown by the application of a modified TNFacytokine that
upregulatesadhesionmolecules,buttheneveneradicatessolid
tumors through rapid destruction of the tumor neovasculature
(349). Enhanced adhesion, e.g., involving ICAM-1 and VCAM-1
up-regulation, may be sufficient to allow T cells to get access to the
primary lymphoma site in the deep parenchyma. Finally, in a time
window to be defined, application of ICB might then unleash the
activity of effector T cells that already invaded the tumor site.
Collectively, efforts to target tumor cells only or single
lymphoma-promoting cellular stromal elements in the TME are
unlikely to confer long lasting remissions. For example, although
anti-CD19 CAR T-cell therapies have proven remarkable efficacy in
B cell malignancies, they become ineffective due to CD19 antigen
loss or downregulation (350,351). Other contributing factors to
A
B
C
FIGURE 3 | Therapeutic strategies to induce vessel normalization and revert endothelial anergy in B-NHL. (A) Anti-angiogenesis therapy tageted at VEGFR-2 or
VEGFR-3 can restore a normalized vessel network. (B) Targeting of the LTbR with LTa
1
b
2
and LIGHT expressing DCs, agonistic antibodies or recombinant factors
potentially circumvents impaired lymphocyte homing by establishing or stabilizing HEV integrity within the lymphoma TME (338). (C) Vessel anergy can be changed
by a targeted conversion of the endothelium toward a reactive endothelium using inflammatory cytokines, which might be site directed to avoid unintended systemic
effects. Normalization of aberrant vessels and activation of the endothelium can also be achieved by locally applied low-dose gamma irradiation (339). Reactive
endothelium within LNs is a prerequisite for an effective infiltration of effector T cells during cellular immunotherapy.
Menzel et al. Angiogenesis in Lymphoma
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substantial rates of treatment failure might be the nodal
immunosuppressive microenvironments in B-NHL (9,352).What
is needed is an integrative concept that blocks vicious feedback
cycles in lymphoma. We suggest that combinatorial targeting of
aberrantly polarized myeloid cell populations, blood endothelial
activation, angiogenesis, and effector T cell dysfunction is a rational
stepwise strategy (Figure 3). Advanced CAR T cell technologies try
to integrate a few of these demands, for example by deleting the
functionality of PD-1 (353,354) and secretion of immune-
stimulatory cytokines such as IL-12, IL-21, or IL-18 (355–357).
We envision that the vasculature is important for control of
lymphoma relapse. In this process, mutual stimulation of residual
tumor cells, mesenchymal and hematopoietic stromal cells, and
endothelial cells might favor neo-angiogenesis and eventually, re-
shaping a growth supporting niche for lymphoma B cells.
AUTHOR CONTRIBUTIONS
LM: conceived the general idea, wrote the manuscript, and
created the figures and tables. AR: conceived the general idea,
co-wrote the manuscript, and edited figures and tables. UH:
provided expert opinion/knowledge input and edited
manuscript, figures, and tables. All authors contributed to the
article and approved the submitted version.
FUNDING
This work was funded by the Wilhelm Sander-Stiftung (grant
number 213.100.02), and by the Deutsche Krebshilfe (grant
number 107749) awarded to AR and UH.
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Conflict of Interest: The authors declare that the research was conducted in the
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potential conflict of interest.
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