Journal of

Clinical Medicine

Review
Cellular and Molecular Players in the Tumor Microenvironment
of Renal Cell Carcinoma
Francesco Lasorsa 1 , Monica Rutigliano 1 , Martina Milella 1 , Matteo Ferro 2 , Savio Domenico Pandolfo 3 ,
Felice Crocetto 3 , Octavian Sabin Tataru 4 , Riccardo Autorino 5 , Michele Battaglia 1 , Pasquale Ditonno 1
and Giuseppe Lucarelli 1, *

1 Urology, Andrology and Kidney Transplantation Unit, Department of Precision and Regenerative Medicine
and Ionian Area, University of Bari “Aldo Moro”, 70124 Bari, Italy
2 Division of Urology, European Institute of Oncology, IRCCS, 71013 Milan, Italy
3 Department of Neurosciences and Reproductive Sciences and Odontostomatology, University of Naples
“Federico II”, 80131 Naples, Italy
4 Department of Simulation Applied in Medicine, George Emil Palade University of Medicine, Pharmacy,
Sciences and Technology, 540139 Târgu Mures, , Romania
5 Department of Urology, Rush University Medical Center, Chicago, IL 60612, USA
* Correspondence: giuseppe.lucarelli@inwind.it or giuseppe.lucarelli@uniba.it

Abstract: Globally, clear-cell renal cell carcinoma (ccRCC) represents the most prevalent type of
kidney cancer. Surgery plays a key role in the treatment of this cancer, although one third of patients
are diagnosed with metastatic ccRCC and about 25% of patients will develop a recurrence after
nephrectomy with curative intent. Molecular-target-based agents, such as tyrosine kinase inhibitors
(TKIs) and immune checkpoint inhibitors (ICIs), are recommended for advanced cancers. In addition
to cancer cells, the tumor microenvironment (TME) includes non-malignant cell types embedded in
an altered extracellular matrix (ECM). The evidence confirms that interactions among cancer cells
and TME elements exist and are thought to play crucial roles in the development of cancer, making
them promising therapeutic targets. In the TME, an unfavorable pH, waste product accumulation,
Citation: Lasorsa, F.; Rutigliano, M.; and competition for nutrients between cancer and immune cells may be regarded as further possible
Milella, M.; Ferro, M.; Pandolfo, S.D.; mechanisms of immune escape. To enhance immunotherapies and reduce resistance, it is crucial first
Crocetto, F.; Tataru, O.S.; Autorino, to understand how the immune cells work and interact with cancer and other cancer-associated cells
R.; Battaglia, M.; Ditonno, P.; et al. in such a complex tumor microenvironment.
Cellular and Molecular Players in the
Tumor Microenvironment of Renal Keywords: renal cell carcinoma; tumor microenvironment; angiogenesis; metabolism; therapy
Cell Carcinoma. J. Clin. Med. 2023, 12,
3888. https://doi.org/10.3390/
jcm12123888

Academic Editor: Antonio G. 1. Introduction

Solimando Renal cell carcinoma (RCC) accounts for about 3–5% of all human cancers, and ac-
Received: 12 May 2023 cording to the 2023 American Cancer Society’s estimates, about 81,800 new cases will be
Revised: 2 June 2023 diagnosed in the USA and 14,890 patients will die from this cancer [1]. Globally, clear-cell
Accepted: 5 June 2023 renal cell carcinoma (ccRCC) represents the most prevalent type of kidney cancer. Tran-
Published: 7 June 2023 scriptomic studies have supported the hypothesis that the proximal tubular epithelial cell
(PTEC) is the cell of origin of the ccRCC [2–4]. Because changes in metabolic pathways
contribute to its development, ccRCC is considered a cell metabolism disease [5–8]. In
ccRCC, cancer cells develop a range of metabolic alterations that support their uncontrolled
Copyright: © 2023 by the authors. growth and proliferation. One such alteration is the activation of the hypoxia-inducible
Licensee MDPI, Basel, Switzerland.
factors (HIFs) pathways, which increase glucose uptake and alter the cellular metabolism
This article is an open access article
to produce energy in an oxygen-independent manner [9–13]. RCC cells also display an
distributed under the terms and
altered lipid metabolism, which is characterized by an increased uptake of fatty acids and
conditions of the Creative Commons
significant accumulations of polyunsaturated fatty acids [14,15].
Attribution (CC BY) license (https://
Surgery plays a key role in the treatment of this tumor, although one third of patients
creativecommons.org/licenses/by/
are diagnosed with metastatic ccRCC and about 25% of patients will develop a recurrence
4.0/).

J. Clin. Med. 2023, 12, 3888. https://doi.org/10.3390/jcm12123888 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2023, 12, 3888 2 of 16

after nephrectomy with curative intent [16–19]. In this scenario, it is urgent to identify novel
biomarkers not only for diagnostic purposes but also for prognostic and predictive factors
of response to therapy [20–24]. Furthermore, the recent introduction of imaging techniques
based on artificial intelligence algorithms will be of considerable support for the risk
stratification, treatment selection, follow-up strategy, and prognosis of this tumor [25–27].
Molecular-target-based agents, such as tyrosine kinase inhibitors (TKIs) and immune
checkpoint inhibitors (ICIs), are recommended for advanced cancers. However, a het-
erogeneous tumor microenvironment (TME) may promote resistance to these systemic
therapies [28,29]. We describe the key characteristics of ccRCC TME in this review to offer
potential directions for future therapeutic approaches.

2. Tumor Microenvironment
In addition to cancer cells, the tumor microenvironment (TME) includes non-malignant
cell types embedded in an altered extracellular matrix (ECM). The composition of the TME
varies between tumor types, but common features include a variety of cells (fibroblasts,
adipocytes, neurons, endothelial cells, immune cells, and stem cells) and secreted molecules
(cytokines, chemokines, growth factors, etc.). Deeper cataloging and comprehension of this
context have been allowed because of novel techniques such as single-cell transcriptomic
sequencing. Over the past years, different studies have linked patients’ prognoses and
therapy responses to the RCC TME composition. The evidence confirms that interactions
among the cancer cells and TME elements exist and are thought to play crucial roles in
the development of cancer, making them promising therapeutic targets [30]. Hence, by
producing growth factors or cytokines and by altering the TME (hypoxia and necrosis),
RCC cells may promote non-tumor cells’ attraction and activation [31].

2.1. Cancer-Associated Fibroblasts (CAFs)

Different theories have been proposed for the origin of cancer-associated fibroblasts
(CAFs). They are described to be less abundant in RCC than in other solid cancers. Trans-
forming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), IL-1, IL-6, and
TNF-α seem to be involved in their recruitment. Resident fibroblasts may give rise to
CAFs. Large amounts of TGF-β are then released by the CAFs, thus initiating an autocrine
signaling loop. In vitro and in vivo studies have already suggested that CAFs may also
arise from adipose-derived stem cells (ASCs), endothelial cells, cancer epithelial cells (as
a result of epithelial-to-mesenchymal transition, or EMT), and bone marrow mesenchy-
mal stem cells (MSCs) [32]. The aberrant expression of smooth muscle actin (α-SMA),
fibroblast-specific protein-1 (FSP1 or S100A4), vimentin, desmin, platelet-derived growth
factor receptor (PDGFR)-α and -β, and fibroblast-activation protein-α (FAP) characterize
these cells. Vascular endothelial growth factor (VEGF), PDGF, TGF-β, epidermal growth
factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), stromal-
derived factor-1α, and osteopontin are known to be secreted by CAFs in the TME. The
extracellular matrix (ECM) is a non-cellular structural component of the TME. Laminin,
fibronectin, collagen type IV, nest protein, and proteoglycan are just a few of the elements
that make up this structure in RCC. By serving as a substrate for cell adhesion and motility
and as a reservoir for the sequestration of released molecules, the ECM promotes inter-
cellular communication in the TME. Preclinical investigations have demonstrated that
CAFs directly inhibit T-cell recruitment or activation by secreting CXCL12 and TGF-β or by
creating a physical barrier through the deposition of ECM. Therefore, they are linked to
T-cell dysfunction and exclusion [33,34]. CAFs may secrete galectin-1 (Gal1), which was
noted to provoke CD8 T cells’ apoptosis. In gastric cancer, Gal1 has also been reported
to promote EMT [35]. Moreover, antitumor immunity may be interfered with indirectly
because immunosuppressive myeloid cells and T regs may be recruited by secreted media-
tors (i.e., IL-6, IL-1 β, etc.). CAFs have a role in tumor cell metabolic reprogramming, EMT
induction, survival pathways, cancer invasion and metastasization, angiogenesis, drug
resistance, immunomodulation, and cytokine secretion (Figure 1).
 cell dysfunction and exclusion [33,34]. CAFs may secrete galectin-1 (Gal1), which was
noted to provoke CD8 T cells’ apoptosis. In gastric cancer, Gal1 has also been reported to
promote EMT [35]. Moreover, antitumor immunity may be interfered with indirectly be-
cause immunosuppressive myeloid cells and T regs may be recruited by secreted media-
tors (i.e., IL-6, IL-1 β, etc.). CAFs have a role in tumor cell metabolic reprogramming, EMT
J. Clin. Med. 2023, 12, 3888 3 of 16
induction, survival pathways, cancer invasion and metastasization, angiogenesis, drug re-
sistance, immunomodulation, and cytokine secretion (Figure 1).

Invasion and
metastasis

ECM remodeling

Immuno-modulation and
cytokines secretion
Angiogenesis
T-cell

B-cell

Cancer-
associated
fibroblast Cancer cell

Drug resistance Metabolic

reprogramming

Figure
Figure1.1.Summary
Summaryofofcancer-associated
cancer-associatedfibroblast
fibroblastbiological
biologicalfunctions.
functions.

An
Anincreased
increasedexpression
expressionofofthe thegenes
genesinvolved
involvedininthe theEMT
EMTpathway
pathwayisisdescribed
describedinin
locallyinvasive
locally invasiveccRCC
ccRCCbecause
becauseofofCAFs
CAFscausing
causingan anECM
ECMremodeling
remodelingwithinwithinthethelesions
lesions
andthen
and thenfacilitating
facilitatingthe
thetumor
tumorspreading.
spreading.Previous
Previousstudies
studieshave
havedemonstrated
demonstratedthe theCAFs’
CAFs’
heterogeneitywithin
heterogeneity withinthethetumor
tumorbulkbulkinindifferent
differentcancer
cancertypes:
types:i.e.,
i.e.,myofibroblasts
myofibroblasts(my- (my-
CAFs),inflammatory
CAFs), inflammatoryCAFS CAFS(iCAFs),
(iCAFs),and
andantigen-presenting
antigen-presentingCAFs CAFs(ap-CAFs)
(ap-CAFs)[36,37].
[36,37].The
The
epithelial-to-mesenchymaltransition
epithelial-to-mesenchymal transitionisisreferred
referredtotoasasaareversible
reversibleprocess
processby bywhich
whichfully
fully
differentiated cells
differentiated cells lose their
theirepithelial
epithelialfeatures
featuresand anddevelop
develop a migratory
a migratory mesenchymal
mesenchymal phe-
notype. Upregulation of ZEB1, ZEB2, Snail, Twist, and Slug leads to E-cadherin
phenotype. Upregulation of ZEB1, ZEB2, Snail, Twist, and Slug leads to E-cadherin loss, loss, which
is considered
which a crucialastep
is considered during
crucial stepEMT. The c-MET/MAPK,
during EMT. The c-MET/MAPK, Wnt/β-catenin, PI3K/AKT,
Wnt/β-catenin,
and JAK/STAT
PI3K/AKT, pathways have
and JAK/STAT been shown
pathways have beento drive
shownmesenchymal traits in RCC.traits
to drive mesenchymal Someinof
these Some
RCC. signaling pathways
of these depend
signaling on growth
pathways factoronreceptors.
depend So, as in
growth factor breast and
receptors. So,pancre-
as in
atic cancer, MUC1 is involved in EMT because it suppresses E-cadherin
breast and pancreatic cancer, MUC1 is involved in EMT because it suppresses E-cadherin expression [38,39].
Moreover, [38,39].
expression EMT initiates the sarcomatoid
Moreover, EMT initiatesconversion of ccRCC,
the sarcomatoid which is characterized
conversion of ccRCC, which by E-
is characterized by E- to N-cadherin switching, membrane dissociation of β-catenin, andof
to N-cadherin switching, membrane dissociation of β-catenin, and enhanced expression
Snail andexpression
enhanced Sparc [40]. of Snail and Sparc [40].

2.2. Tumor Vascular Cells

Lower levels of adhesion molecules are expressed by the tumor endothelial cells
(TECs), thus impairing the barrier function as does the reduced interaction between the
TECs and pericytes. Pericytes also interact with other stromal cells and cancer cells,
modulating the TME [41]. Tumor blood vessels are notably characterized by irregular
branching, tortuous course, arteriovenous shunting, and an altered surface area to volume
ratio [42]. Leaky and disorganized tumor vessels also affect cell oxygenation and immune
J. Clin. Med. 2023, 12, 3888 4 of 16

cell dysfunction, and reduce drug penetration. Upon binding to its receptor (VEGF 1-
2-3), VEGF activates downstream messengers, which lead to the expression of genes
responsible for the proliferation, survival, migration, and permeability of the vascular
endothelial cells. VEGFR is coupled to an intracellular tyrosine or serine/threonine kinase.
mTOR is an essential part of the PI3K/AKT signaling system, which controls several
biological processes such as protein synthesis, angiogenesis, and autophagy. Deregulation
of mTOR signaling is related to the development of cancer [43]. Tumor-associated myeloid
cells (i.e., neutrophils and macrophages) may enhance angiogenesis via pro-angiogenic
mediators, including VEGF, FGF2, PIGF, and BV8. The RCC cells recruit mast cells and
cancer endothelial cells through modulating the PI3K/AKT/GSHβ/AM signaling [44].
Cytogenetic abnormalities (aneuploidy) have been described in TECs in RCC. In association
with high glycolytic activity, this finding reflects a hyperactivated phenotype, although
TECs have always been thought not to be able to proliferate [45]. In addition, the androgen
receptor (AR) may promote angiogenesis by recruiting endothelial cells in RCC via the
AKT/NF-kB/CXCL5 axis [46]. Lymphatic endothelial cells (LECs) cover the walls of
lymphatic vessels, representing a dissemination route for cancer cells. TECs and LECs
may express immune checkpoint molecules such as PD-L1 (programmed-death-ligand-1),
IDO1, and TIM3. At the same time, LECs may present tumor antigens in the absence of
co-stimulatory signals. For these reasons, LECs and TECs have been recognized as possible
regulators of antitumor immunity and immunotherapy response [47,48].

2.3. Tumor-Associated Adipocytes

The surrounding adipose microenvironment may regulate the activity of tumor and
non-tumor renal epithelial cells. Adipocytes have been reported to release free fatty
acids, hormones, cytokines, adipokines, and growth factors, which may impact cancer
progression [49,50]. Therefore, they may promote a pro-tumorigenic low-grade chronic
inflammation [51]. Adiponectin gene polymorphism rs182052 is associated with ccRCC
risk, and leptin receptor gene polymorphism rs1137101 may also be a possible risk factor
for RCC [52,53]. Robust glycogen and lipid accumulation is observed in ccRCC. Lipid
droplets store cholesterol esters and triglycerides within the cancer cells. Recently, Ferrando
et al. compared human adipose explants from normal (hRAN) and kidney cancer (hRAT)
tissue. A higher expression of leptin and its receptor (ObR) and smaller adipocytes were
noted in hRAT than in hRAN. These findings may relate to increased lipolysis and therefore
increased energy availability in hRAT. Because leptin is known to induce a fibroblastoid
morphology in breast cancer, it is speculated that it may contribute to the upregulation of
EMT markers in RCC [54,55].

2.4. Tumor Immune Microenvironment

NK cells, effector T cells, and mature dendritic cells are tumor-associated immune
cells that may be involved in the anticancer immune response, whereas regulatory T cells
and myeloid-derived suppressor cells (MDSCs) have the opposite impact.

2.4.1. T Cells
Antigen-presenting cells (APCs) such as dendritic cells and their major histocompat-
ibility complex (MHC) are necessary for T cells’ activation. The T-cell receptors (TCR)
recognize the antigen peptides in MHC; CD8 T cells bind class I MHC, whereas CD4 T
cells bind class II MHC. The T-cell coreceptors CD4 and CD8 bind the non-polymorphic
domains of MHC. However, co-stimulation is required to fully activate effector T cells:
CD28 binds B7-1 (CD80) or B7-2 (CD86) on APCs or B cells. CD8 T cells destroy their
targets via granzyme, perforin-mediated apoptosis, or via the FAS-FASL axis [56]. CD4
helper T cells affect a variety of other immune cells. According to their phenotype, CD4
T cells may exert dual effects. For instance, the Th1 subtype enhances CD8 T cells and
B cells, and it may directly kill cancer cells via IFN-γ or TNF-α. On the opposite side,
the Th2 subtype releases anti-inflammatory mediators, thus limiting antitumor responses.
J. Clin. Med. 2023, 12, 3888 5 of 16

Immune checkpoint molecules represent pivotal elements involved in the cancer immune
escape. PD-L1 on the renal epithelial cancer cells binds T cells’ inhibitory receptor PD-1
(programmed death-1). Upon binding, the activated T cells either die or lose their func-
tion. Another PD-1 ligand, PD-L2 (also known as B7-DC), is known to be expressed by
tumor-infiltrating dendritic cells. Activated T cells may express CTLA-4 (cytotoxic T lym-
phocyte antigen-4), which provides negative feedback signals for T-cell activation at the
lymph node level. Other immune checkpoint molecules (B7-H3, B7-H4, VISTA, PD-1H,
TIM-3, LAG-3, TIGIT, etc.) have been identified, and clinical studies have evaluated their
clinical relevance [57–59]. A continuous transition to terminally exhausted clonotypes
has been found using transcriptome analysis of CD8 T cells in ccRCC. Indeed, naïve, cy-
totoxic, exhausted, progenitor, and terminally exhausted T cells have been isolated. In
advanced and metastatic ccRCC microenvironments, higher exhausted T cells with low
TCR diversity were inferred than in normal kidney samples or the peripheral blood [60,61].
Giraldo et al. investigated the associations among the infiltration of CD8 T cells and mature
dendritic cells (DCs), the expression of immune checkpoint molecules, and the patients’
prognosis. A poor prognosis characterized the first group, with a strong expression of
immune checkpoint molecules and low mature DCs. In turn, mature DCs and a lower
expression of immune checkpoint molecules were associated with a better prognosis [62].
The same group then identified three immune profiles of ccRCC: immune-regulated (CD8
PD-1+TIM-3+LAG-3+ TILs and T regs), immune-activated (CD8 PD-1+ TIM-3+ TILs), and
immune-silent (enriched with TILs similar to those in the adjacent non-malignant tissue).
Remarkably, the immune-regulated tumors had a significant risk of disease progression
and had aggressive histologic features [63]. FoxP3 regulatory T cells (T regs) are a subpop-
ulation of CD4 T cells with immunosuppressive properties in the TME. These cells work in
the healthy host to promote immunological homeostasis and self-tolerance. In turn, these
cells contribute to suppressing effective antitumor immunity via different mechanisms,
including the expression of cytokines (IL-2, IL-10, TGF-β, adenosine), direct cytotoxicity
(perforin and granzyme), promotion of tolerogenic dendritic cells with a reduced capacity
to activate effector T cells, and augmentation of T-reg production [64–66]. Immune check-
point molecules (PD-1, CTLA-4, Tim-3, LAG3, TIGIT, etc.) may be expressed by FoxP3 T
regs to further limit anticancer responses. As a consequence, the CD8 T-cell population
increases and tumor growth slows upon reducing the T-reg population in the TME [67].

2.4.2. Tumor-Associated Myeloid Cells

Tumor-associated macrophages (TAMs) may arise from different sources, such as
tissue-resident macrophages or bone-marrow-derived infiltrating ones. Macrophages and
neutrophils are phagocytic effectors of anticancer innate immunity. The simple binary
states of classical versus alternative activation were first used to categorize myeloid cells.
Macrophage polarization not only depends on intrinsic signaling (ERK, NF-kB, and STAT1
vs. STAT3 and STAT6 pathways) but it is also regulated by immune, stromal, and cancer
cells in the TME in a context-dependent manner [68]. M1 polarization depends on Th1
cytokines (IFN-γ); M1 macrophages release pro-inflammatory cytokines such as IL-6,
IL-12, IL-23, and TNF-α and toxic compounds (i.e., reactive oxygen species-ROS). Th2
cytokines (IL-4, IL-10, and IL-13) promote M2 macrophages, which are known to reduce
inflammation and promote angiogenesis, wound healing, and tissue remodeling. M2-TAMs
are typically characterized by an impaired antigen presentation but an increased expression
of angiogenic factors (VEGF), tissue remodeling metalloproteases (MMPs), cathepsins,
TGF-β, IL-10, prostaglandin E2, and other molecules that may limit lymphocyte and
macrophage proliferation and function. Therefore, after a tumor has developed, M2-TAMs
may dampen immune surveillance and alter the ECM to accelerate tumor growth [69].
Similar to TANs, immune checkpoint molecules may be expressed by TAMs, which may
also use T regs to further suppress antitumor immunity [70]. Upon binding to signal
regulatory protein-α (SIRP-α, an inhibitory receptor on phagocytes), CD47 on tumor cells
may block phagocytosis. Worse prognoses and more aggressive phenotypes of ccRCC
J. Clin. Med. 2023, 12, 3888 6 of 16

have recently been linked to CD47 expression [71,72]. Multiple TAM phenotypes have
been described, and different TAM subsets may coexist within tumors [73]. Different
transcriptomic patterns are displayed by the macrophage subgroups among the different
tumor types. This supports the concept that tumor-associated myeloid cells are imprinted
according to the organ and cancer type. In ccRCC specimens, a continuum from M1-like to
M2-like states was shown by the TAM populations. It is well established that TAMs are
involved in different processes of tumorigenesis, ranging from initiation to angiogenesis,
metastasis, and immune escape. CCL2 may recruit macrophages to hypoxic regions of
the tumors, where increased HIF1α/HIF2α induces the transcription factor of several
angiogenesis-related genes. Here, the TAMs may release growth factors, cytokines, MMPs,
and other molecules that promote blood vessel formation and stabilization [74]. Hence,
a high CD68+ TAM density has been associated with a high microvessel density [75].
Previous studies have reported that a higher number of TAMs in the TME is associated
with poorer prognoses and earlier relapses in RCC patients [76,77]. Chittezhath et al.
showed that IL-1-IL-1R signaling is crucial for controlling the tumor-promoting phenotypes
of the monocytes and macrophages in RCC. Therefore, further investigations are required
to uncover the potential therapeutic role of anti-IL-1 for selected human cancers [78].
Tumor-associated neutrophils (TANs) are also categorized as N1 (antitumor) or N2
(pro-tumor), depending on their effects. Direct or antibody-dependent cytotoxicity, along
with the stimulation of several innate and adaptive immune cells (NK cells, B and T
cells, and DCs), are the main mechanisms through which N1 action against tumors is
exhibited [79]. In a murine model of RCC, N1-TANs were shown to build an antimetastatic
barrier, thus limiting cancer spreading to the lungs [80]. In turn, N2-TANs may promote,
directly or indirectly, tumor growth, angiogenesis, and metastasis. Several cytokines have
been shown to guide neutrophils’ recruitment in mice and in human solid cancers. IFN-γ
has been noted to stimulate N1 polarization, whereas TGF-β promotes N2-TANs [81]. A
high neutrophil to lymphocyte ratio (NLR) in both the peripheral blood and the TANs is
linked to a poor prognosis in RCC patients [82]. Intriguingly, Song et al. found that N2-
TANs may promote the progression of RCC via the androgen receptor/c-Myc pathway [83].
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous group of myel-
oid cells [84]. According to their origin, from granulocytic or monocytic myeloid cell
lineages, granulocytic/polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs
(M-MDSCs) are the two main categories of MDSCs in humans and mice. Pro-inflammatory
mediators (prostaglandin E2, IL-6, VEGF, and complement fragment C5a) and growth
factors (GM-CSF, M-CSF) are demanded for their recruitment and activation from the bone
marrow at tumor sites. While M-MDSCs use nitric oxide (NO) and immunosuppressive
cytokines (IL-10 and TGF-β), as well as the expression of immune checkpoint molecules
such as PD-L1, PMN-MDSCs preferentially use reactive oxygen species (ROS), peroxynitrite,
and prostaglandin E2 (PGE2) to mediate immune suppression. Additionally, MDSCs
may enhance cancer immune escape via the deprivation of essential amino acids such as
cysteine, arginine (Arg), and tryptophan (TRP) because they may express arginase-1 and
indolamine 2,3-dioxygenase 1 (IDO1) [85–87]. MDSCs may also play a crucial role in the
formation of the premetastatic niche. The chemokine receptors CXCR2 and CXCR4 are
primarily responsible for attracting neutrophils or PMN-MDSCs to the premetastatic niches.
By inhibiting immune cells, inducing ECM remodeling, and angiogenesis, MDSCs may
facilitate the engraftment of tumor cells in the premetastatic niche [88].

3. Metabolic Reprogramming and Immune Escape in the TME

Large amounts of energy are required for tumor cells’ growth, proliferation, and
metastasis. Oncogenic signals affect the metabolic pathways in cancer cells: increased gly-
colysis, glutaminolysis, and lipolysis support bioenergetic demands. Increased utilization
of glutamine is often found in ccRCC to generate citrate and lipids. Indeed, glutamine,
cysteine, or glutamate deprivation may be beneficial for the treatment of Von Hippel–
Lindau (VHL)-deficient RCC [89]. Despite oxygen availability, they typically show aerobic
J. Clin. Med. 2023, 12, 3888 7 of 16

glycolysis (the Warburg effect), which is responsible for TME acidification because of lactate
accumulation. Lactate may suppress the activation of effector T cells and limit the differ-
entiation of monocytes and DCs, whereas it promotes T regs and the M2-like phenotype
of TAMs [90,91]. An unfavorable pH, waste product accumulation, and competition for
nutrients between cancer and immune cells may be regarded as further possible mecha-
nisms of immune escape [92,93]. Nutrients’ availability in the TME also depends both on
systemic (the patient’s diet and nutritional state) and local factors (the tumor type and its
location within the primary tissue) [94,95]. pVHL loss and HIF1α stabilization promote
the expression of glycolytic transporters and enzymes such as GLUT1, hexokinase 1 (HK1)
and 2 (HK2), pyruvate kinase muscle isozyme 2 (PKM2), pyruvate dehydrogenase kinase
1 (PDHK1), and lactate dehydrogenase A (LDHA). A metabolomic analysis of ccRCC
revealed different distributions of intermediates between the upper and lower parts of the
glycolytic flux. A significant reduction in metabolites of the lower chain was noted because
metabolites are rerouted toward the pentose phosphate pathway (PPP). NADH dehydro-
genase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) inhibits Complex I of the
electron transport chain (ETC). It is significantly overexpressed in ccRCC, as it is under the
control of HIF1α. Mitochondrial dysfunction is thought to be a hallmark of cancer cells.
Abnormal mitochondrial numbers and morphology; dysfunctional ETC; mitochondrial
DNA (mtDNA) mutations; and oxidative damage to lipids, proteins, and nucleic acids are
some of their distinguishing features. Several studies have noted the crosstalk between
HIF1α accumulation and mitochondrial dysfunction in different cancer types. HIF1α
limits triglyceride lipase-mediated lipolysis by HIG2, thus reducing fatty acid oxidation.
Additionally, HIF1α delays ETC via NDUFA4L2, COX4-2, Complex I, and Complex IV [96].
Decreased cell viability, increased cisplatin susceptibility, inhibition of autophagic machin-
ery, increased mitochondrial mass, and ROS accumulation were found after silencing or
knocking down NDUFA4L2 [12]. The process of autophagy, which involves the fusion
of vesicles (autophagosomes) with lysosomes with hydrolytic enzymes, allows cells to
degrade and recycle proteins and organelles. Autophagy may be promoted by starvation
and oxidative stress conditions to sustain metabolic demands; therefore, it may sustain
cancer cells’ growth. When selective for mitochondria, it is referred to as mitophagy. It
is well established that such a complex process involves different proteins (such as the
autophagy-related Atg proteins) and that it modulates interactions between cancer cells and
non-cancer cells in the TME. Two different pathways of mitophagy have been unveiled so
far: PINK1/Parkin (depending on membrane depolarization) and BNIP3/NIX/FUNDC1
(depending on hypoxia). Of note, HIF1α upregulates BNIP and NIX expression [97–99].
In the TME, tumor cells may compete with CD8 T cells for different amino acids such
as arginine, tryptophan, serine, cysteine, and alanine. Increased uptake of arginine by
cancer cells, and its consumption by TAMs (via arginase-1), reduce its availability in the
TME. Reduced mTORC activity in the T cells results in reduced T cells’ effector functions
and increased memory-like T cells [100,101]. Extracellular serine has been demonstrated
to be essential for the growth and effector capabilities of T cells, which are compromised
when serine levels are low in the TME [102].
Three metabolic pathways consume tryptophan (TRP): protein synthesis, serotonin,
and kynurenine (KYN) production [103]. Two enzymes are known to transform TRP to
KYN: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1). The
expression of IDO1 may also be induced by TNF-α and IFN-γ. Riesenberg et al. explored
the role of IDO1 expression in TECs in ccRCC. They identified an increased microvascular
density in tumors with higher IDO+-TECs [104]. Chen et al. reported the upregulation of
TDO in CAFs in renal cancer [105]. In addition to TRP depletion in the TME, the tumorigenic
function of KYN appears to be mediated by its interaction with aryl hydrocarbon receptors
(AhR) on immune and cancer cells. The KYN/AhR axis may facilitate cancer cells’ survival,
migration, and chemoresistance. Immunosuppressive T reg cells are differentiated because
of the activation of AhR in CD4 T cells [106]. Additionally, PD-1 expression on CD8 T cells is
induced by KYN [107]. Recently, the authors have investigated the role of MUC1 in the TME
J. Clin. Med. 2023, 12, 3888 8 of 16

of ccRCC. In MUC1H tumors, M2-like TAMs (CD68+CD163+) are able to produce KYN.
Moreover, MUC1H samples have shown increased deposition of C1q, which colocalized
with pentraxin-3 (PTX3), in association with higher expression of proangiogenic receptors
(C3aR and C5aR). PTX3 is known to activate the classical cascade of the complement
system [108,109]. Nonetheless, the increased expression of CD59 limited C5b-9 assembly in
the TME of MUC1H ccRCC. Finally, this study demonstrated a lower expression of PD-L1
in MUC1H samples [110].

4. Clinical Role of the TME and Therapeutic Implications

The balance of pro- and anti-angiogenic signals that regulates angiogenesis is referred
to as the “angiogenic switch.” Pro-angiogenic signals (VEGF-A, the FGF receptor family,
and MMPs) are counteracted by anti-angiogenic factors such as thrombospondin 1 and 2,
angiopoietin, endostatin, osteopontin, angiostatin, and cellular communication network
factor 3 (CCN3) [111]. Anti-angiogenic signals are overcome because angiogenesis is a
defining characteristic of malignancies. Somatic mutations of the Von Hippel–Lindau (VHL)
gene are observed in approximately 92% of patients diagnosed with ccRCC. VHL loss leads
to a constitutive activation of hypoxia-induced response elements (HRE), genes involved in
metabolism (GLUT1, PDK1, and EPO), proliferation, cell survival, and angiogenesis (VEGF
and PDGF). Because of its crucial role, anti-angiogenic therapies have been developed for
the treatment of patients with advanced clear-cell and non-clear-cell RCCs [112].

4.1. Angiogenesis Inhibitors

Bevacizumab is a recombinant humanized monoclonal antibody that prevents circu-
lating VEGF from binding to its receptor on the endothelial cell surface [113,114]. In 2003,
it showed superiority as a single agent in metastatic ccRCC compared with placebo. Then,
its use in combination with IFNα was approved in a metastatic setting. This combination
is recommended by the European Society of Medical Oncology (ESMO) guidelines in
metastatic ccRCC patients with a good or intermediate prognosis [115]. Subsequently,
different oral anti-angiogenic tyrosine kinase inhibitors (TKI) have been introduced for the
treatment of advanced RCC. TKIs have different targets and several sites of action. For
instance, sunitinib blocks VEGFR and PDGFR tyrosine kinases, as well as FMS-like tyrosine
kinase 3 (Flt-3), colony-stimulating factor 1 receptor (CSFR1), and neurotrophic factor.
These tyrosine kinases affect not only angiogenesis, but also tumor growth and metastatic
progression. In a phase III study, sunitinib overcame IFNα-2a in terms of progression-free
survival (PFS), objective response rate (ORR), and quality of life (QoL) [116,117]. Pazopanib
targets VEGFR, FGFR, PDGFR, and c-Kit, limiting angiogenesis and tumor growth [118].
In a phase III clinical trial, it demonstrated PFS and ORR benefits over placebo, so that
pazopanib has been approved as a first-line therapy for metastatic ccRCC [119]. Sorafenib
is active against VEGFR, PDGFR, c-Kit, Flt-3, and RET-receptor kinases, thus decreasing
angiogenesis and cell replication. However, when tested against placebo in a phase III
study, it demonstrated benefits in PFS but not overall survival (OS). Therefore, it has been
approved for the treatment of advanced ccRCC patients who failed prior INFα or IL-2
therapy [120]. Axitinib is a second-generation TKI against VEGFR that has been approved
for metastatic ccRCC when prior sunitinib or cytokine treatment has failed [121,122]. In
a phase IIII study, cabozantinib provided a better PFS and ORR than everolimus in the
CABOSUN trial when compared with sunitinib. Several cabozantinib targets are known
so far (VEGFR2, MET, ROS1, TYRO3, Flt-3, c-Kit, RET, AXL, etc.), which are involved in
cancer progression at different levels [123]. It has been approved as a first-line therapy
for metastatic ccRCC patients with poor or intermediate prognosis and as second-line
treatment in case of prior failed VEGF-targeted therapies [124].

4.2. Mammalian Target of Rapamycin (mTOR) Inhibitors

As mentioned above, mTOR plays a crucial role in the PI3K/AKT axis, which controls
angiogenesis, cell proliferation, and metabolism. Additionally, HIF expression is also pro-
J. Clin. Med. 2023, 12, 3888 9 of 16

moted by mTOR. Therefore, mTOR inhibition has been introduced as a target in RCC [125].
Due to their superior efficacy and tolerability, various targeted and ICI treatments have
replaced it in clinical practice. Temsirolimus was approved by the EMA as a single drug for
the first-line treatment of adult patients with at least three out of six negative prognostic
factors according to the MSKCC classification [126]. On the basis of the RECORD I research,
everolimus was approved for use in the treatment of ccRCC that had progressed after
receiving first-line therapy [127].

4.3. Cytokine Therapy and Immune Checkpoint Inhibitors (ICIs)

Although chemoresistant, RCCs have long been thought to be highly immunogenic.
Spontaneous remissions of metastatic ccRCC patients after surgery were observed in the
1960s. Immunotherapy drugs enhance the host’s antitumor immune responses rather than
directly destroying their targets. IL-2 and IFN-α were the initial immunotherapy regimens
used to treat metastatic RCC. A better understanding of immune escape mechanisms
led to the development of antibodies against immune checkpoint molecules, which are
currently used for advanced RCCs. Ipilimumab (anti-CTLA-4) was the first ICI to be
introduced for the treatment of metastatic ccRCCs [128]. When compared with everolimus,
nivolumab (anti-PD-1) had superior tolerability and an improved OS and ORR [129].
However, the combination of ipilimumab and nivolumab has shown promising efficacy
and greater response rates than either agent alone. Over time, clinical trials have evaluated
the role of pembrolizumab (anti-PD-1), atezolizumab, avelumab, and spartalizumab (PD-L1
inhibitors) as therapeutic agents for ccRCC. The efficacy of PD-L1 as a prognostic marker for
mccRCC is still debated, even though elevated PD-L1 expression appears to be predictive
of responsiveness to checkpoint inhibitors. PD-L1 positive patients seem to respond better
to anti-PD-1/PD-L1 agents than PD-L1 negative patients, although both groups benefit
from ICI when compared with the sunitinib group [130]. Preclinical research has shown
that angiogenic inhibition can increase T-cell infiltration into tumors, increasing the efficacy
of ICI [131]. This rationale paved the way to phase III studies exploring this combination
approach (Table 1) [132–136]. Recent European Association of Urology (EAU) guidelines
consider ICI and TKI combination therapy as a first-line treatment for metastatic RCC.
The International Metastatic RCC Database Consortium (IMDC) scale is applied to stratify
patients according to their predicted prognosis [137].

Table 1. Phase III clinical trials evaluating therapeutic combinations of immune checkpoint inhibitors
and anti-angiogenic agents for advanced-stage ccRCC. PFS: progression-free survival; OS: overall
survival.

Trial Drugs Primary Endpoint

NCT02420821 Atezolizumab + Bevacizumab PFS [132]
NCT02853331 Pembrolizumab + Axitinib PFS, OS [133]
NCT02684006 Avelumab + Axitinib PFS, OS [134]
NCT02811861 Pembrolizumab + Lenvatinib PFS [135]
NCT03141177 Nivolumab + Cabozantinib PFS [136]

Finally, belzutifan, the first HIF inhibitor, has been approved for use in advanced
ccRCC with VHL disease, and further studies are evaluating its clinical efficacy in associa-
tion with ICI and other targeted therapies [138].

5. Conclusions
A significant number of patients diagnosed with advanced-stage RCC remain un-
responsive or even develop resistance to the currently available systemic therapies. To
enhance immunotherapies and reduce resistance, it is crucial to first understand how the
immune cells work and interact with the cancer and other cancer-associated cells in such
a complex tumor microenvironment. Exploring the different metabolic pathways in the
TME may give novel approaches to reduce immune suppression and to limit metastasis.
J. Clin. Med. 2023, 12, 3888 10 of 16

Developing novel predictive biomarkers, adopting the optimal therapeutic regimens, or

combining them in accordance with risk models might be useful to improve survival
outcomes, therapeutic safety, and quality of life of RCC patients.

Author Contributions: Conceptualization, F.L. and G.L.; methodology, F.L.; software, G.L.; valida-
tion, F.L. and G.L.; formal analysis, F.L.; investigation, F.L. and G.L.; resources, G.L.; data curation,
F.L., M.R., M.M., M.F., S.D.P., F.C., O.S.T., R.A., M.B., P.D. and G.L.; writing—original draft prepara-
tion, F.L., M.R., M.M., M.F., S.D.P., F.C., O.S.T., R.A., M.B., P.D. and G.L.; writing—review and editing,
G.L.; visualization, F.L.; supervision, G.L.; project administration, G.L. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This study did not require ethical approval.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created.
Conflicts of Interest: The authors declare no conflict of interest.

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