Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 839-852 © Pleiades Publishing, Ltd., 2024.

839

REVIEW

Tumor-Associated Senescent Macrophages, Their Markers,

and Their Role in Tumor Microenvironment

Tamara V. Pukhalskaia

1,2,3

, Taisiya R. Yurakova

, Daria A. Bogdanova

1,3

and Oleg N. Demidov

1,3,4,a

Sirius University of Science and Technology, 354340 Federal Territory Sirius, Sirius Russia

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia

INSERM UMR1231, Université de Bourgogne, 21000 Dijon, France

e-mail: Oleg.Demidov@u-bourgogne.fr

Received December 21, 2023

Revised April 27, 2024

Accepted April 27, 2024

Abstract— Tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment

(TME) and the most abundant population of immune cells infiltrating a tumor. TAMs can largely determine direc-

tion of anti-tumor immune response by promoting it or, conversely, contribute to formation of an immunosup-

pressive TME that allows tumors to evade immune control. Through interactions with tumor cells or other cells in

the microenvironment and, as a result of action of anti-cancer therapy, macrophages can enter senescence. In this

review, we have attempted to summarize information available in the literature on the role of senescent macro-

phages in tumors. With the recent development of senolytic therapeutic strategies aimed at removing senescent

cells from an organism, it seems important to discuss functions of the senescent macrophages and potential role

of the senolytic drugs in reprogramming TAMs to enhance anti-tumor immune response and improve efficacy

ofcancer treatment.

DOI: 10.1134/S0006297924050055

Keywords: senescent cells, p16

INK4

, p21

cip1

, CD206, CXCR1, tumor microenvironment, immunosuppression

Abbreviations: Arg1,arginase; BMDM,bone marrow-derived macrophages; LCCM-BMDM,bone marrow-derived macrophages

cultivated in L929-cell conditioned medium; M-CSF-BMDM, bone marrow-derived macrophages cultivated with recombi-

nant M-CSF; NAD,nicotinamide adenine dinucleotide; SCA,single-cell analysis; TME,tumor microenvironment; SASP,senes-

cence-associated secretory phenotype; TAMs,Tumor-associated macrophages; TGF-β,transforming growth factor beta.

* To whom correspondence should be addressed.

INTRODUCTION

Tumor microenvironment (TME) comprises a

complex heterogenous system including immune cells,

endothelial and stromal cells, as well as extracellular

matrix [1-7]. Recent analysis of single-cell transcrip-

tome (single-cell analysis, SCA) demonstrated that up

to 90% of cells in the tumor microenvironment could

be non-transformed [8]. TME composition varies de-

pending on the type and stage of the tumor devel-

opment; organ in which a primary tumor emerges;

factors expressed by the tumor cells, and patient’s an-

amnesis [9].

Recently more attention has been paid to the ef-

fects of cell aging, or the so-called senescence state on

the tumor growth and development [10]. Phenotype of

senescent cell is heterogenous and depends on the type

of the cells subjected to senescence and factors causing

this state. In general, senescence is defined by the irre-

versible arrest of cell cycle, elevated lysosomal activi-

ty, resistance to apoptotic stimuli, enhanced glycolysis,

increased DNA damage, as well as by the increased

secretion of chemokines, cytokines, and growth fac-

tors combined under the name senescence-associated

secretory phenotype (SASP). Through the paracrine

effects on the surrounding cells, SASP could facilitate

PUKHALSKAIA et al.840

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

initiation of epithelial-mesenchymal transition [11, 12],

acquiring pluripotency (stemness) by the cells [13, 14],

local tissue invasion [15], angiogenesis [16], fibroblast

activation [17], immunosuppression [18, 19], enhanced

metastasizing [20], and resistance to therapy [21].

Based on the above-mentioned functions, identi-

fication of the senescent cells most often is based on

such markers as enhanced expression of cell cycle in-

hibitors, p16

INK4

and p21

cip1

[22]; increased activity of

lysosomal β-galactosidase associated with aging [23];

increased content, in comparison with other cells, of

the phosphorylated form of H2AX histone reflecting

presence of DNA damage; cytokines typical of SASP,

such as, IL-6 [24]. It must be mentioned that at present

there is no unique single marker of senescence, and

most often several methods are used to confirm this

cell phenotype.

In addition to spontaneous aging, the therapy-in-

duced aging is often detected in TME. It was shown in

various studies that the classic cytotoxic therapy, tar-

geted therapy, and immunotherapy could induce cell

aging [25]. Under the action of these factors all cell

types in TME could be subjected to senescence and af-

fect tumor cells. However, the tumor-associated mac-

rophages (TAMs), which play an important role in TME

comprising the most prevalent population of immune

cells infiltrating tumor, attract most attention in the

context of senescence [26]. As immune system cells,

they define, to a large extent, immune landscape of the

TME, and could facilitate the disease progression [8].

In particular, it was shown that the presence of TAMs

in a tumor is associated with poor prognosis of the dis-

ease and low efficiency of therapy [27-29]. Despite the

fact that removal or reprogramming of senescent TAMs

seems as a promising approach to increase efficiency

of cancer therapy, up to recent times information on

the phenotype typical for the senescent macrophages

in TME and mechanism underlying its formation was

practically absent. Hence, it seems reasonable to dis-

cuss progress in the area of research associated with

the active search for universal biomarkers of the se-

nescent TAMs and with the recently introduced novel

models for detection of biological effects of this cell

subpopulation on the tumor growth and development.

SENESCENT MACROPHAGES IN A TUMOR

Same as other types of cells in tumor microenvi-

ronment, TAMs could be subjected to senescence, how-

ever, potential biological mechanisms of appearance

of senescent macrophages in a tumor and their prog-

nostic value are poorly understood [28]. The fact that

the in  vivo and in  vitro phenotypes of the macrophages

from the same type of tumor could differ significantly

complicates investigation of this issue. Moreover, the

heterogeneity of the mechanisms and pleiotropy of

the senescence mediators in tumor diseases is further

complicated by the genetic diversity of human tumors

and complex interactions between the tumor cells and

cells in their microenvironment during oncogenesis

[30]. At the same time, cells in TME could also have

a very heterogenous phenotype. In particular, macro-

phages in  vivo are characterized by the wide spectrum

of subpopulations, in which subpopulation of senes-

cent macrophages seems to be therapeutically signifi-

cant. The features of senescent macrophages allowing

to distinguish them from other subpopulations of mac-

rophages will be described below (figure).

Phenotype of senescent TAMs. To date the fol-

lowing subpopulations of TAMs have been described

the most: proinflammatory M1-like and immunosup-

pressive M2-like macrophages [more detailed descrip-

tion of M1 and M2 macrophages are provided in the

paper by Yurakova etal. [31] in this issue of Biochem-

istry (Moscow) journal]. Phenotype of the senescent

macrophages in the tumor microenvironment could

be assigned neither to the traditional polarization

classes typical for M2 (high expression of Arg1), nor

typical for M1 (high expression of iNOS). Macrophages

in TME could have different phenotypes depending of

the tumor type, nature of therapy, and particular clin-

ical manifestations [32]. Hence, search for the specific

markers allowing selective distinguishing of the senes-

cent macrophages from the other TAMs is an import-

ant task for the development of therapy targeting this

population of the cells. Available data and identified

markers typical for the senescent and M1/M2 macro-

phages are summarized and compared in Table1 [33].

Aging is accompanied by chronic inflammation,

which is manifested by the increase of expression of

SASP components: proinflammatory cytokines IL-1α,

IL-6, and TNF, C-reactive protein, matrix metallopro-

teases MMP-3 and MMP-9 modulating extracellular

matrix, as well as chemokines CXCL-1 and CXCL-10 at-

tracting neutrophils and monocytes, respectively [46,

54]. All these factors could affect tumor cells and their

environment. It was concluded in the recent meta-re-

view by Moss etal. that the markers of proinflamma-

tory (M1) macrophages including CD11c, iNOS, MHC-II,

and CD80 commonly increase with age [55]; however,

it is still unknown how this is associated with the pat-

tern of tumor microenvironment.

Another part of the data is concentrated on the

fact that the senescent macrophages have a M2-like

phenotype. It is generally recognized that the macro-

phages with M2-polarization demonstrate enhanced

expression of arginase-1 (Arg1) involved in arginine

metabolism [33], and facilitate development of the

non-small-cell lung cancer, and their presence in a tu-

mor microenvironment correlates with lower survival

of the patients, increased metastasizing, and enhanced

TUMOR-ASSOCIATED SENESCENT MACROPHAGES 841

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Potential markers of senescent TAMs. More universal markers found in different models of tumor genesis are shown on the

left; and characterized TAMs from the lung tumors are shown on the right. Combined increase of the expression of inhibitors of

cyclin-dependent kinases p16

INK4

and/or p21

cip1

, Arg1, CD206, CXCR1, CD38 is typical for the senescent TAMs, as well as increased

arginine metabolism and glycolysis. SASP of the senescent TAMs is characterized by secreted molecules such as TGF-β, IL-10,

IL-6, TNF, IL-1β, MMP12, TIMP2, CXCL12, CXCL13, HGF. For the lung cancer models the following molecules secreted by the

TAMs were detected: Bmp2, Ccl2, Ccl7, Ccl8, Ccl24, Cxcl13, and Il10. The markers Abca1, Fizz1, Rack1, Mcp-1, Cd40, Cox2 have

been less investigated, but have certain diagnostics potential for identification of senescent macrophages. Created with the

help ofBioRender.com.

Table 1. Comparison of the markers typical for M1/M2 and senescent macrophages

Markers

[34-37]

M2a

[8, 34, 37, 38]

M2b

[38, 39]

M2c

[38, 40]

M2d

[38, 41-43]

Senescent macrophages

[8, 44-53]

TNF, IL-6 + – + – – +

IL-10 – + + + + +

TGF-β – + – + – +

IL-1 + – + – – +

CD206 – + – – – +

ARG1 – + – + + +

iNOS + + – – + +/–

MHC-II, CD80 + – – – – +/–

CD163 – + + – – +

CD38 + – – – – +

Expression of Toll-like

receptors (TLRs)

TLR2/4 – – TLR1/8 –

expression

of all TLRs decreases

PUKHALSKAIA et al.842

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

proliferation of tumor cells [56, 57]. Moreover, the ag-

ing TAMs also exhibit the M2-like immunosuppressive

phenotype, which was demonstrated in several studies

[8, 34, 37-43]. Gene clusters expressed in the senescent

macrophages from the samples derived from the pa-

tients with refractory bladder cancer were identified

using single-cell transcriptome analysis [27]. Among

those genes, the gene of transforming growth factor

beta (TGF-β) was detected, which is characteristic for

the immunosuppressive tumor microenvironment. Ex-

pression of the aldo-keto reductase B gene (AKR1B1)

was also noted, which plays an important role in in-

flammation and metabolism of various chemotherapy

preparation, as well as in cell differentiation, prolifer-

ation, and apoptosis [28, 58].

Both in humans and in mice the senescent TAMs

demonstrate reduced expression of the molecules of

major histocompatibility complex (MHCII) and of the

Toll-like receptors (TLR) [52], which is accompanied by

the decrease of phagocytic activity [59], efferocytosis,

and autophagy [46]. This also characterizes the aging

macrophages as more immunosuppressive cells de-

spite the enhanced expression of SASP.

New potential markers of the senescent macro-

phages (ABCA1, FIZZ1, RACK1, MCP-1, CD40, COX2) re-

ported in the recent systematic review by Moss etal.

[55] should be also mentioned. It is likely that these

markers also play an important role in the senes-

cence of TAMs. Despite the fact that there is no unique

marker identifying aging cells, overexpression of the

cyclin-dependent kinases p16

INK4a

and p21

cip1

has been

used for a long time as a marker of aging cells invitro

and in  vivo both in mouse and human models [48, 60,

61] including for macrophages [49, 50, 62].

Metabolism of senescent TAMs. In addition to

phenotypic characteristics, functional and metabolic

features of the senescent macrophages also should be

considered. One of the peculiarities of the senescent

cells is preservation of metabolic activity supporting

SASP program and other specific functions despite the

arrest of cell cycle [63]. As a rule, accumulation of dys-

functional mitochondria occurs in the senescent im-

mune calls, as well as activation of glycolysis instead

of oxidative phosphorylation (OXPHOS), which leads to

bioenergetic imbalance [64].

Similar metabolic adaptations are typical for the

proinflammatory M1 macrophages, which predominate-

ly use glycolysis and demonstrate reduced OXPHOS,

while the alternatively activated M2 macrophages de-

pend mostly on OXPHOS [65, 66]. From the functional

point of view enhanced glucose metabolism in TAMs

is required for synthesis of various molecules involved

in SASP, in particular, it facilitates enhanced secretion

of cathepsin-B by macrophages and tumor progression

[63, 67, 68]. Interestingly enough, metabolic adapta-

tions of other types of senescent cells could be differ-

ent from the adaptations in the senescent TAMs. For

example, increase of oxidative phosphorylation is typi-

cal for the senescent endothelial cells [69], hepatocytes

[70, 71], β-cells in diabetes [72, 73], and hematopoietic

stem cells [74]. However, increase of glycolysis is more

typical for the senescent macrophages. Inhibition of

this metabolic pathway could potentially be an ap-

proach for senolytic therapy [75].

Nicotinamide adenine dinucleotide (NAD) is pres-

ent in an organism in an oxidized (NAD

) and reduced

state (NADH). NAD is an important coenzyme for re-

dox reactions, which plays one of the key roles in the

energy metabolism [76], and also is a substrate for sir-

tuins, PARP1, and ectoenzymes CD38 and CD157 [77].

Numerous studies have shown that NAD

levels de-

crease with age [78]. It has also been shown that the

monocytes and macrophages of elderly patients have

reduced respiratory capacity due to insufficient level

of NAD [64].

Activation of CD38 during aging could lead to in-

creased signalling by NF-κB in macrophages [53, 79],

which facilitates induction of the SASP factors ex-

pression [80]. The SASP factors, such as IL-6 secreted

in the tumor microenvironment, could activate the

expression of CD38 in macrophages [63]. The ectoen-

zymes CD38 and CD157, in turn, can hydrolyse NAD

in tumour tissues and release extracellular adenosine,

which is involved in immunosuppression [81]. Hence,

aging cells expressing CD38 could initiate loss of NAD

which could lead to an increase of the number of se-

nescent cells through the effects of SASP factors via

the positive feedback mechanism.

The NF-κB signaling and the level of mitochondri-

al Ca

(mCa

) play an important role in regulation of

inflammatory response, induction of the SASP pheno-

type, and polarization of macrophages [82]. Association

between the NF-κB signaling and calcium metabolism

was observed during analysis of 700 human transcrip-

tomes. This analysis revealed that age correlates with

the expression of the mitochondrial calcium uniporter

gene (MCU) and its regulatory subunit MICU1. These

genes play an important role in the transmission of

mCa

signals [47].

EFFECTS OF SENESCENT TAMs

ON TUMOR PROGRESSION

Pathogenic effects of senescent macrophages on

growth and development of tumors in the non-small

cell lung cancer models were demonstrated in the re-

cent year. Haston et al. established a mouse model,

p16-FDR, which allowed to establish that TAMs and,

to a lesser degree, endothelial cells comprise the main

pool of senescent cells populating the KRAS-induced

lung tumors [8]. Removal of the p16

INK4a+

senescent

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BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Table 2. Main senolytics currently used

Senolytic/Senomorphic Mechanism of action Effect on senescent macrophages References

Dasatinib + Quercetin

inhibition of Src/Abl

HIF-1α, PI3-kinase

exerts effects [84, 85]

ABT-263 (Navitoclax) inhibition of proteins of Bcl-2 family exerts effects [86]

ABT-737 inhibition of proteins of Bcl-2 family exerts effects [8]

Venetoclax proteins of Bcl-2 family not investigated [87]

Fisetin PTEN-mTor cascade exerts effects [88]

SR12343 inhibitor of NF-Kb not investigated [89, 90]

Apigenin IRAK1/IκBα/NF-κB not investigated [91, 92]

Cardiac glycosides mainly inhibitors of Na

-ATPase not investigated [93-95]

cells using senolytic approaches was shown to pro-

long survival and inhibit tumor growth [8, 83]. Senes-

cent TAMs in the p16-FDR model demonstrated the

CD206

p16

INK4a+

phenotype and also expressed numer-

ous tumor-stimulating SASP factors, which were found

to be unique to the tumors formed in the lungs (Bmp2,

Ccl2, Ccl7, Ccl8, Ccl24, Cxcl13, and Il10), and not typical

for the previously described classic SASP factors such

as Tnf, Mcp1, Il6, Il1b Il7, Mmp12, Timp2, Cxcl-12/13,

Hgf) [8, 50, 51]. The study by Haston etal. [8] became a

key study in the field of senescent macrophages in tu-

mors, as there was no clear understanding of whether

the macrophages actually had senescent cells proper-

ties. Interestingly enough, the macrophage phenotype

similar to that one observed in the KRAS-induced lung

tumors in mice was also found in the old mice at the

age of 20-22 months.

A previously established transgenic mouse line

(INK-ATTAC) [29] was used in the study by Prieto

et al. Using SCA, the authors found with the help of

SCA a population of senescent alveolar macrophages

SIGLEC

p16

INK4a+

CXCR1

high

localized in the tissues of old

mice and in lung tumors. The SIGLEC

p16

INK4a+

CXCR1

high

subpopulation promoted adenoma formation and in-

hibited proliferation and tumor infiltration with the

CD8

lymphocytes. Targeted depletion of the p16

INK4a+

senescent cells in the INK-ATTAC model prevented de-

velopment of negative effects mediated by the senes-

cent cells in TME, and slowed down oncogenesis in the

KRAS mice [29].

The studies by Prieto etal. [29] and Haston etal.

[8] show that the strategy involving removal of senes-

cent macrophages from the TME has an anti-tumor po-

tential. This approach could be suggested as an adju-

vant therapy for treatment of oncological diseases.

A new class of drugs, the senolytics have been

suggested as agents for selective removal of senescent

cells. Information on the most popular and widely

used senolytics is presented in Table2. Some of them

have already been approved for clinical use in the

treatment of various diseases, hence, so their repur-

posing for senolytic therapy could occur within a short

period of time, and such preparations might be used

for reprogramming of tumor microenvironment in the

cancer patients in the nearest future.

VARIETY OF MODELS FOR INVESTIGATION

OF SENESCENT TAMs

In the final section of the review, we consider im-

portant to summarize existing approaches to model-

ing of TAMs and discuss whether some of the models

could be used to model senescent TAMs. In particular,

the following in  vitro approaches have been suggested

in the literature: association of the primary macro-

phages or cell lines with the tumor adding tumor-cell

conditioned medium [96], co-cultivation, cultivation in

a Transwell® system [33, 97], isolation of TAMs direct-

ly from tumor tissues and their following in  vitro culti-

vation [98-100].

Human TAMs in  vitro. The most popular approach

is based on the use of transformed human monocytic

cell lines such as THP-1 and U937 after stimulation

with phorbol-myristate-acetate. The obtained mac-

rophages are cultivated in a tumor-cell conditioned

medium [101], in some cases factors facilitating de-

velopment of the M2-like phenotype (IL-4 and IL-10)

are added [102]. This approach helps with the model

standardization; however, this approach has several

PUKHALSKAIA et al.844

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

drawbacks for investigation of senescent TAMs such

as initial priming of the cells in the direction of the

M2-like phenotype, which limits the possibilities for

monitoring changes in the transcription and metabolic

profiles in this particular model in response to associa-

tion with tumor and following induction of aging.

Another method for creation of human TAMs is

based on obtaining macrophage precursors from the

human peripheral blood by cultivation in the presence

of human granulocyte-macrophage colony-stimulating

factor (GM-CSF) followed by activation with IFN-g, LPS

for M1, while for obtaining of M2 and TAMs macro-

phages are differentiated in the medium containing

human macrophage colony-stimulating factor (M-CSF)

followed by stimulation with IL-4 [103].

It is worth noting that the 2D in  vitro system, in

which TAMs are prepared by adding tumor-cell con-

ditioned medium directly to macrophages, is often

insufficient to detect changes associated with the se-

nescent macrophages. In particular, only co-cultivation

of macrophages with tumor cells in the study by Enu-

kashvily etal. [104] allowed detecting transcription of

pericentromeric satellite repeats HS2/HS3, which are

likely to be associated with aging. These differences

demonstrate importance of direct intercellular interac-

tions between the cells of microenvironment and tu-

mor cells, which is also important to consider during

selection of the model of TAMs.

Mouse TAMs in  vitro. It is our opinion that the

in  vitro models of human and mice TAMs have a

number of fundamental differences. In particular,

the main approach to generate mouse TAMs in  vitro

is based on the use of bone marrow-derived macro-

phages (BMDM). Two possible methods of macrophage

differentiation for modelling mouse TAMs have been

described for mouse TAMs modeling in the litera-

ture: (i) by addition of mouse M-CSF (M-CSF-BMDM);

(ii)by addition of a medium conditioned by the L929

(LCCM) cell line (LCCM-BMDM) [31]. In addition to

M-CSF, LCCM contains chemerin (Rarres2), factor in-

hibiting migration of macrophages (Mif), osteopontin,

Ccl7, Ccl2, Cxcl1, Cx3cl1, Ccl9, Gerem1, and Tgf-β [105].

Macrophages generated by these two methods differ

in a number of parameters, which has been described

previously; the differences were observed both in the

case of stimulation with LPS and in the non-activat-

ed state. The LCCM-BMDM secrete Tnf, Il-6, and Il-12

at lower levels after LPS stimulation compared to the

M-CSF-BMDM, show increase of glycolysis indicators,

have larger mitochondrial mass with high percent of

dysfunctional mitochondria. Secretion of Il-10 by the

non-activated LCCM-BMDM has been demonstrated in

comparison to M-CSF-BMDM [106]. This has to be tak-

en in consideration when working with the senescent

TAMs models in  vitro, as the increase of the Il10 ex-

pression in macrophages in the course of inflammato-

ry processes has been described as one of the changes

associated with aging [55].

The models investigating interactions of TAMs

with tumor microenvironment in  vitro provide sev-

eral clear advantages despite being artificial to a cer-

tain degree. In particular, this approach enables less

labor-consuming experimental design, is low-cost, and

provides the possibility to perform high throughput

screening of large libraries of chemical compounds

with the goal of identification of novel medicinal

preparations allowing to decrease negative properties

of TAMs and to increase their anti-tumor activity.

In recent years investigations of tumor microen-

vironment using cultivation of tumor organoids have

been actively developing. This approach occupies an

intermediate position between the studies of the role

of TAMs in  vitro and in  vivo; while this approach re-

mains relatively simple and cost-effective, it allows ef-

fective modeling of complex three-dimensional inter-

actions of TAMs with extracellular matrix and various

types of cells in TME [107]. Considering rapid progress

in this area, it could be expected that the experimen-

tal protocols involving cultivation of organoids will

become very popular in the nearest future.

Study of TAMs in  vivo. Experimental laboratory

animal models of oncogenesis using predominantly

mouse models remain the gold standard for the study

of TAMs. Supplemented with success in single-cell se-

quencing (single-cell analysis, SCA) and SCA data on

different signatures of TAMs derived ex  vivo from the

biopsies of cancer patients, this approach allows re-

ceiving most accurate results and expand our knowl-

edge on the role of macrophages at different stages of

oncogenesis [108-111]. In the section devoted to the ef-

fects of senescent TAMs on tumor progression, success-

ful examples of creating and using in  vivo models to

study senescent TAMs have been presented [29]. Data

on characteristics of these mouse models and a num-

ber of other promising in  vivo

models for the study of

senescence are summarized in Table  3. Information

presented in the Table  3 could help the interested re-

searchers with selection of an appropriate experimen-

tal model [8, 29, 48, 62, 98-100, 112, 113].

CONCLUSIONS

Acquiring senescent phenotype by the cells of tu-

mor environment including TAMs could significantly

affect tumor progression and its resistance to mod-

ern anti-tumor therapies. Senescent modality of TAMs

does not totally fit to the presently existing function-

al classification of macrophages such as M1/M2 po-

larization. Number of studies devoted to analysis of

tumor microenvironment at the level of single cells

is increasing exponentially; furthermore, novel and

TUMOR-ASSOCIATED SENESCENT MACROPHAGES 845

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Table 3. Mouse models for investigating senescence

Name

of the mouse

model

Description

Reporter system

(color of senescent cells)

System

for removal

of senescent cells

Inducibility

of the system

References

p16-3MR

BAC transgene;

reporter cassette

under control

of p16

INK4a

promoter

luciferase/mRFP

(red, ex584/em607)

HSV-TK +

+ ganciclovir

only cell

removal –

ganciclovir

[114]

p16-Cre and

p16-CREERt2

knock-in

of the cassette

into the last exon

p16

INK4a

after

STOP codon

Rosa26-mTmG

(green, ex484/em510)

Rosa26-lsl-DTA

Tamoxifen

for p16-CREERt2

[62]

p16Ink4a-

CreERT2

knock-in

of the CreERT2 gene

into the first exon

of p16

INK4a

Rosa26-CAG-lsl-tdTomato

(orange, ex554/em581)

Rosa26-SA-lsl-

DTR-IRES-

tdTomato

Tamoxifen;

Tamoxifen +

+ diphtheria

toxin

[115]

p21-Cre

attP transgene;

reporter cassette

under control

of the minimal

promoter

p21

cip1

p21-CreERT2-

IRES-GFP

Rosa26-CAG-lsl-tdTomato

(orange, ex554/em581);

or (green, ex484/em510)

Rosa26-lsl-LUC/

Rosa26-lsl-DTA

Tamoxifen [48]

INK-ATTAC

transgene;

reporter cassette

under control

of the minimal

promoter p16

INK4a

FKBP–Casp8-IRES-EGFP

(green, ex488/em507)

FKBP–Casp8

only cell

removal –

AP20187

[29, 116]

p16-FDR

knock-in

of the cassette into

the last exon

p16

INK4a

after

the STOP codon;

P16-P2A-FLPo-

P2A-DTR-mCherry

Rosa26 frt-STOP-frt-EGFP

(green, ex488/em507)

or DTR-mCherry

(red, ex587/em610)

DTR-mCherry

only cell

removal –

diphtheria

toxin

[8]

Note. HSV-TK, herpes simple virus thymidine kinase gene; DTA, diphtheria toxin A gene; DTR, diphtheria toxin receptor

gene; LPo, optimized flippase-recombinase gene; mTmG, loxP-tdTomato-STOP-loxP-GFP; FKBP–Casp8–FK506-binding-protein

caspase8 gene.

improved in  vivo models for investigation of senes-

cent TAMs have been suggested in recent years. All

this would facilitate detailed characterization of this

subpopulation of myeloid cells in tumors and expand

our understanding of the role of senescent TAMs in

oncogenesis in the nearest future. We hope that the

progress in methodology of investigation of senescent

TAMs discussed in this review will help the readers to

select appropriate strategy in investigation of various

aspects of tumor growth. Senescent TAMs, as a sepa-

rate population of cells, deserves special attention of

the researchers; the recently introduced new class of

therapeutic preparations, senolytics, which limit nega-

tive effects of the senescent cells by changing the frac-

tion of senescent cells in the tumor microenvironment,

could increase significantly efficiency of various anti-

cancer therapies.

Acknowledgments. The authors are grateful to

Drutskaya Marina Sergeevna for valuable discussions

and advices.

Contributions. T.V.P., O.N.D., conceptualization;

T.V.P., D.A.B., wrote the manuscript; T.V.P., D.A.B., visu-

alization; O.N.D., D.A.B., T.R.U., T.V.P., edited the manu-

script. All authors have read and agreed to the pub-

lished version of the manuscript.

PUKHALSKAIA et al.846

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Funding. This work was financially supported by

the Russian Science Foundation (grant 19-75-20128).

Work of T. V. Pukhalskaia and D. A.  Bogdanova was

in part supported by the Ministry of Science and High-

er Education of the Russian Federation (Agreement

no.075-10-2021-093; Project NIR-IMB-2102).

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

Theauthors of this work declare that they have nocon-

flicts of interest.

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