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Clinical Application Perspectives of Lung Cancers 3D Tumor Microenvironment Models for In Vitro Cultures

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Despite the enormous progress and development of modern therapies, lung cancer remains one of the most common causes of death among men and women. The key element in the development of new anti-cancer drugs is proper planning of the preclinical research phase. The most adequate basic research exemplary for cancer study are 3D tumor microenvironment in vitro models, which allow us to avoid the use of animal models and ensure replicable culture condition. However, the question tormenting the scientist is how to choose the best tool for tumor microenvironment research, especially for extremely heterogenous lung cancer cases. In the presented review we are focused to explain the key factors of lung cancer biology, its microenvironment, and clinical gaps related to different therapies. The review summarized the most important strategies for in vitro culture models mimicking the tumor–tumor microenvironmental interaction, as well as all advantages and disadvantages were depicted. This knowledge could facilitate the right decision to designate proper pre-clinical in vitro study, based on available analytical tools and technical capabilities, to obtain more reliable and personalized results for faster introduction them into the future clinical trials.
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Citation: Wieleba, I.;
Wojas-Krawczyk, K.; Krawczyk, P.;
Milanowski, J. Clinical Application
Perspectives of Lung Cancers 3D
Tumor Microenvironment Models for
In Vitro Cultures. Int. J. Mol. Sci.
2022,23, 2261.
https://doi.org/10.3390/
ijms23042261
Academic Editors: Isotta Chimenti
and Roberto Gaetani
Received: 31 December 2021
Accepted: 16 February 2022
Published: 18 February 2022
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International Journal of
Molecular Sciences
Review
Clinical Application Perspectives of Lung Cancers 3D Tumor
Microenvironment Models for In Vitro Cultures
Irena Wieleba * , Kamila Wojas-Krawczyk , Paweł Krawczyk and Janusz Milanowski
Pneumonology, Oncology and Allergology Department, Medical University of Lublin, 20-059 Lublin, Poland;
kamilawojas@wp.pl (K.W.-K.); krapa@poczta.onet.pl (P.K.); janusz.milanowski@umlub.pl (J.M.)
*Correspondence: i.m.wieleba@gmail.com
Abstract:
Despite the enormous progress and development of modern therapies, lung cancer remains
one of the most common causes of death among men and women. The key element in the develop-
ment of new anti-cancer drugs is proper planning of the preclinical research phase. The most adequate
basic research exemplary for cancer study are 3D tumor microenvironment
in vitro
models, which
allow us to avoid the use of animal models and ensure replicable culture condition. However, the
question tormenting the scientist is how to choose the best tool for tumor microenvironment research,
especially for extremely heterogenous lung cancer cases. In the presented review we are focused
to explain the key factors of lung cancer biology, its microenvironment, and clinical gaps related
to different therapies. The review summarized the most important strategies for
in vitro
culture
models mimicking the tumor–tumor microenvironmental interaction, as well as all advantages and
disadvantages were depicted. This knowledge could facilitate the right decision to designate proper
pre-clinical
in vitro
study, based on available analytical tools and technical capabilities, to obtain
more reliable and personalized results for faster introduction them into the future clinical trials.
Keywords: lung cancer; tumor microenvironment; organoids; spheroid; tumor-on-chip
1. Introduction
Cancer is a leading disease in mortality worldwide. Tremendous progress in cancer
diagnostic and therapies has been made in the past years. Tumorigenesis consists of three
main stages: initiation, promotion, and progression. During initiation and promotion, nor-
mal cells change their phenotype, based on anchored genetical mutations, into a cancerous
one. One may observe increasing metabolomic, intensive cell differentiation within changes
in tumor stroma mechanics and increased cell mobility. In the progression stage interaction
between tumor cells, stromal cells and immune cells provide to neovascularization and
metastasis initiation [
1
,
2
]. During the epithelial–mesenchymal transformation (EMT) cancer
cells develop a mechanism of immunosurveillance and drug resistance. For better analysis
of this process in an
in vitro
model, it is required to use tumor microenvironment (TME)
elements together with cancer cells. One significant technical aspect, which cannot be
solved in classical 2D
in vitro
cell culturing, is tumor heterogenicity. Next, interaction be-
tween tumor and its stromal cells is investigated, as well as interaction between tumor and
tumor infiltrated immune cells. Cell-to-cell interactions are regulated by specific molecular
pathways. This type of interaction and their input on tumor progression cannot be clearly
examined during cell culturing in monolayer [
3
5
]. The golden mean of 3D culturing
technics is a precision imaging on tumor spatial organization ex vivo. The development
process for new therapeutic or new diagnostic methods starts from basial research. The key
point to gain more applicable preclinical studies is the selection of the most appropriate
analytical tool, which will give us the possibility to applicate conducted data into clinical
practice. The aim of this paper is to summarize the main directions in three-dimensional
(3D) culturing technologies with the aim of solving challenges from lung cancer diagnosis
and treatment.
Int. J. Mol. Sci. 2022,23, 2261. https://doi.org/10.3390/ijms23042261 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 2261 2 of 24
Lung cancer (LC) is a cancer type leading in morbidity and mortality rate worldwide.
There are about 11 genetically differentiated and 2 main histopathological subtypes of
lung cancer. Non-small cell lung cancer (NSCLC) refers to 85% of all lung cancer cases.
Another 15% are classified as small-cell lung cancer (SCLC), which is more aggressive in
the metastatic stage [
6
]. Therapeutical options for lung cancer treatment include surgery,
stereotaxic radiotherapy, chemotherapy, targeted therapy, and immunotherapy. A major
percentage of LC are diagnosed in the advanced disease stage. The main therapeuti-
cal strategy for non-operative patients in the IIIB–IV disease stages is a combination of
chemotherapy and targeted or immunotherapy. For patients with driver mutations, such as
EGFR (10% of NSCLC cases) and BRAF (0.5–4.9% of lung adenocarcinomas), and rearrange-
ments in ROS1 (1–2% of lung adenocarcinomas) and ALK (3–7% of lung adenocarcinomas)
gene-targeted therapy can be used [
6
,
7
]. Nowadays, the main immune therapy strategy
used for LC treatment is immune checkpoint inhibitors (ICIs). The introduction of ICI
therapy into clinical practice gave a significant elongation in progression-free survival
time and overall survival time, but the data are not highly satisfactory. High tumor muta-
tional burden, microsatellite instability, and programmed death ligand 1 (PD-L1)-positive
expression are used as predictor factors for ICI immune therapy and are still under inves-
tigation [
8
]. Nevertheless, in standard clinical practice, only PD-L1 expression on cancer
cells with immunohistochemistry staining is examined and used as a validated predictive
factor. According to clinical data, this marker does not have a high prediction sensitivity or
successful translation into the effectiveness of the therapy [
9
14
]. Research searching for
clinically appliable predictor factors is highly needed. One significant problem is the high
frequency of adverse effects in the III and IV degree of severity through patients treated
by ICI [
15
17
]. More specific analysis of cancer–microenvironment and immunity–drug
interactions may give knowledge on how to improve clinical benefits from treatment.
As mentioned above, lung cancer is highly heterogenous. Mutation diversity profile
is linked with the presence of driver mutations. EGFR-positive tumors include a higher
percentage of branch mutations (different from mainly occurred somatic mutations) than
trunk mutations (commonly present in all somatic tissue). Circulating tumor DNA (ctDNA)
encodes trunk mutations in general and does not reflect tumor heterogeneity [
18
]. Single-
cell transcriptomic analysis of biopsy samples collected from patients with NSCLC in the
III/IV disease stage showed significant differentiation in cellular regulation processes,
molecular tumor development, and cell phenotype. Several scientific groups instigated
studies concerning the changes in dependency quality composition of immune cells in
TME according to disease stage [
19
21
]. On the basis of reports about high variability
of tumors’ genetic, molecular, and phenotyping profiles amongst patients with the same
histopathological cancer type, there is still a necessity to uncover the mechanisms of
interaction between tumors and TME’s compartments, improving highly precise basial
research with the potential for use in clinical practice [2224].
For the analysis of drugs, the most appropriate oncological model is the one that
approximates the changes occurring at an advanced stage of the disease. However, in order
to learn about new mechanisms of the neoplastic process, all stages of the disease may
be considered. Nevertheless, 2D cultures give far less reliable results, even in studies on
primary tumors, because they do not retain the native form of intercellular contact. Main
strategies for
in vitro
study on tumor–tumor microenvironment interactions involved the
use of tumor spheroids/organoids and patient-derived xenografts. Thorough analysis
of pre-clinical models of NSCLC were already published [
25
,
26
]. Here, we analyze the
main challenges in the field of lung cancer research, taking into account the key features of
tumor–microenvironment interactions and technical possibilities for providing
in vitro
3D
tumor microenvironments for lung cancer cultures.
Int. J. Mol. Sci. 2022,23, 2261 3 of 24
2. Lung Cancer and Its Tumor Microenvironment
2.1. Lung Cancer Origin
Lung cancer is classified in two main subgroups: non-small cell lung cancer (NSCLC)
and small-cell lung cancer (SCLC). Every histopathological subtype of lung cancer has a
specific localization and different metastatic properties. The representative characteristic of
lung cancer subtypes is presented in Figure 1. About 40% of all cases is classified as lung
adenocarcinoma. For this histology type, more common genetic alternations include EGFR,
BRAF,ROS1,RET,PRKCB,NTRK,MET,HER2, and ALK, as well as somatic mutations in
KEAP1 and STK11. For squamous cell lung cancer (SQCLC), mutations generally occur in
PRKCA,PKN1,FGF,FGFR1,FGF3,MYC,CDKN2A,RB1,TP53,CDKN2A,NOTCH1,MLL2,
and NFE2L2 genes [
6
,
27
]. High tumor mutation burden (TMB) occurs in smokers, which
is predicted by long-time exposure on different tumorigenic substances from cigarettes.
Clinical sample analysis showed differences not only in somatic mutations type and level,
but also in epigenetic regulation, mRNA and miRNA expression, and protein level.
Kim et al. used single cell sequencing of patient-derived material to study genes expres-
sion dedifferentiation in different tumor associated cell types based on tumor localization
and disease stage. They observed inequal intratumor gene progression levels in accordance
with tumor progression. They also analyzed different subtypes of epithelial cells, stromal,
and immune cells and matched single-cell phenotypes with the metastatic process. Based
on the collected data, Kim et al. proposed twelve fibroblast subtypes, three subtypes
of alveolar macrophages, and six dendritic cells (DCs) subpopulation in patient-derived
probes in accordance to cell genetic profile [
19
]. Another single cell-sequencing analysis of
lung adenocarcinoma samples defined two main types of intratumor cells: the first similar
to epithelial alveolar cells, the second with upregulation of EGFR and JAK/STAT pathways
and increased stimulation of the EMT process by transforming growth factor
β
(TGF
β
)
and hypoxia-corelated factors [
19
,
20
,
28
]. The key pathways in the epithelial–mesenchymal
transformation process involved TGF
β
, Wnt,
β
-catenin, and VEGFR. The main pathways
connected via interaction between tumor and TME concerns are p53 protein, Myc, Hippo,
and Flower [
29
]. NSCLC is defined by higher tumor mutation burden than SCLC. SCLC is
a neuroendocrine lung cancer subtype quickly metastasized. Low grade neuroendocrine
tumor cells are more adhesive than those with high grade, but both subtypes are more likely
to generate three dimensional structures. SCLC is less heterogenic than NSCLC
[3032].
Herein, in accordance to the presence of major transcriptional factors, five phenotype
subtypes of SCLC were investigated: SCLC-A (ASCL1), SCLC-N (NEUROD1), SCLC-P
(POU2F3), SCLC-Y (YAP1), and SCLC-I (ASCL1-/NEUROD1-/POU2F3-) [
33
]. Transcrip-
tomic analysis of patient-derived samples defined combined PLCG-2- positive phenotype,
corelated with higher disease aggressiveness in patients with SCLC [
34
]. Apart from pheno-
typically differentiated neoplastic cells, there are also cancer stem cells (CSCs). The origin
of cancer stem cells depends on the tumor localization in the lungs. In the SQCLC subtype,
cancer stem cells are derived from surface epithelium stem cells or submucosal gland. In
the SCLC subtype, CSCs develop from Clara cells and pulmonary neuroendocrine cells, and
in lung adenocarcinoma from pulmonary stem cell or pneumocytes comprehensively. Lung
cancer stem cells typically express EpCAM, CD44, CD90, and CXCR-4. CD44-positive and
CXCR4-positive CSCs have the ability to form spatial structures
in vitro
and in xenograft
mice models [
35
]. CSCs are involved in silencing antitumor immune responses through the
production of immunosuppressive and pro-inflammatory cytokines, neovascularization
stimulation, and tumor–stromal interactions by TGF
β
production. In lung adenocarcinoma,
CSCs are involved in immune therapy resistance. Gene profile for LUAD CSCs are better
known than for SCLC [
36
]. CSCs’ induced treatment resistance involves ALDH1,CD44,
and PTEN genes, regulated by testis-specific Y-like protein 5 in NSCLC subtypes [
37
]. CSCs
isolated from primary lung cancer samples indicate a correlation between SOX2-positive
and NANOG-positive lung adenocarcinomas with high levels of ALDH. In SCLC samples,
this correlation was adverse. Tumor cells with high ALDH level formed permanent tumor
spheres, while SCLC cells with low expression of ALDH were not able to form spatial cul-
Int. J. Mol. Sci. 2022,23, 2261 4 of 24
ture [
38
]. CSCs are crucial for tumor progression and drug resistance, but there is still a need
for precise methods of obtaining and evaluating their role in cancer diagnostics [
35
,
39
44
].
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 24
for SCLC [36]. CSCs’ induced treatment resistance involves ALDH1, CD44, and PTEN
genes, regulated by testis-specific Y-like protein 5 in NSCLC subtypes [37]. CSCs isolated
from primary lung cancer samples indicate a correlation between SOX2-positive and
NANOG-positive lung adenocarcinomas with high levels of ALDH. In SCLC samples,
this correlation was adverse. Tumor cells with high ALDH level formed permanent tumor
spheres, while SCLC cells with low expression of ALDH were not able to form spatial
culture [38]. CSCs are crucial for tumor progression and drug resistance, but there is still
a need for precise methods of obtaining and evaluating their role in cancer diagnostics
[35,39–44].
Figure 1. Histological types of lung cancer: NSCLC: SQCLC–28%, LUAD–38%, AIS–2%, LCC–10%;
SCLC–15%, LCNEC–5%, and Lung Carcinoid–2% of all cases (this graph was created in GraphPad Prism
9).
2.2. Tumor Stroma
Fast fibrosis and neovascularization process conditioning in the metastasis formation
are associated with higher tumor aggressiveness. Tumor cells are responsible for recruit-
ing stromal cells (myofibroblasts) and immune cells to initiate multi-stage progression.
Fibroblasts are involved in extracellular matrix formation due to the synthesis of struc-
tural proteins, e.g., integrins, collagen, or elastin [45,46]. Cho et al. showed the myofibro-
blast impact on fibronectin remodeling through paracrine communication with tumors
[47]., A fibroblast subtype crucial for tumor progression include cancer associated fibro-
blasts (CAFs) developed from myofibroblasts. CAFs are involved in tumor extracellular
matrix destabilization and the promotion of angiogenesis. Hu et al. defined three func-
tional subtypes of NSCLC-derived CAFs based on the different expression levels, HGF,
FGF7, and p-SMAD2. There was dependency between CAFs functional subtype, targeted
therapy effectiveness (all patients had driver mutations), and patients’ pre-treatment con-
dition. CAFs functional differentiation is regulated among others by TGF-β [48,49]. Hao
et al. indicated two subtypes of CAFs corelated with NSCLC in accordance to desmo-
plastic (low- and high-desmoplastic CAFs). These subtypes showed different regulation
in tumor progression and matrix destabilization [50]. Sato et al. showed correlation be-
tween TGFβ secretion from CAFs and tumor heterogeneity in lung adenocarcinomas.
TGFβ stimulated lung adenocarcinomas tumor plasticity into acinar type [51]. Hence,
TGFβ mediates a certain role in cell-to-cell cross talk between tumor and tumor microen-
vironments. It is also involved in extracellular matrix changes through progression and
initiate bone-metastasis [52]. CAFs’ co-culture use is required to induce tumor plasticity
in 3D models [53,54]. Pericytes interact with TME elements through the production of
chemokines and cytokines, but may also polarize into CAFs within stimulation of tumor
fibrosis [55]. Pericytes take an important role in tumor progression through the formation
of tumor stroma, immune cells regulation, tumor angiogenesis, and neovascularization
development and engagement into defeating the brain–blood barrier through lung cancer
brain metastasis formation. Bichsel et al. excluded lineage-EpCAM-CD73+CD90+ peri-
vascular-like cells from patients’ primary lung cancer samples, with high expression of
Figure 1.
Histological types of lung cancer: NSCLC: SQCLC–28%, LUAD–38%, AIS–2%, LCC–10%;
SCLC–15%, LCNEC–5%, and Lung Carcinoid–2% of all cases (this graph was created in GraphPad Prism 9).
2.2. Tumor Stroma
Fast fibrosis and neovascularization process conditioning in the metastasis formation
are associated with higher tumor aggressiveness. Tumor cells are responsible for recruit-
ing stromal cells (myofibroblasts) and immune cells to initiate multi-stage progression.
Fibroblasts are involved in extracellular matrix formation due to the synthesis of structural
proteins, e.g., integrins, collagen, or elastin [
45
,
46
]. Cho et al. showed the myofibroblast
impact on fibronectin remodeling through paracrine communication with tumors [
47
].
A fibroblast subtype crucial for tumor progression include cancer associated fibroblasts
(CAFs) developed from myofibroblasts. CAFs are involved in tumor extracellular matrix
destabilization and the promotion of angiogenesis. Hu et al. defined three functional
subtypes of NSCLC-derived CAFs based on the different expression levels, HGF, FGF7, and
p-SMAD2. There was dependency between CAFs functional subtype, targeted therapy ef-
fectiveness (all patients had driver mutations), and patients’ pre-treatment condition. CAFs
functional differentiation is regulated among others by TGF-
β
[
48
,
49
]. Hao et al. indicated
two subtypes of CAFs corelated with NSCLC in accordance to desmoplastic (low- and high-
desmoplastic CAFs). These subtypes showed different regulation in tumor progression and
matrix destabilization [
50
]. Sato et al. showed correlation between TGF
β
secretion from
CAFs and tumor heterogeneity in lung adenocarcinomas. TGF
β
stimulated lung adeno-
carcinomas tumor plasticity into acinar type [
51
]. Hence, TGF
β
mediates a certain role in
cell-to-cell cross talk between tumor and tumor microenvironments. It is also involved in
extracellular matrix changes through progression and initiate bone-metastasis [
52
]. CAFs’
co-culture use is required to induce tumor plasticity in 3D models [
53
,
54
]. Pericytes interact
with TME elements through the production of chemokines and cytokines, but may also
polarize into CAFs within stimulation of tumor fibrosis [
55
]. Pericytes take an important
role in tumor progression through the formation of tumor stroma, immune cells regulation,
tumor angiogenesis, and neovascularization development and engagement into defeating
the brain–blood barrier through lung cancer brain metastasis formation. Bichsel et al.
excluded lineage-EpCAM-CD73
+
CD90
+
perivascular-like cells from patients’ primary lung
cancer samples, with high expression of PD-L1, IL-6, and basal
α
-SMA molecule. Pericytes
from patients were proliferated from mesenchymal stem cells, where mice-model pericytes
were proliferated from epithelium cells [
56
]. Under the influence of VEGF, epithelial hyper-
plasia occurs, the integrity of the blood vessel wall is violated, and a new network of vessels
associated with the tumor develops. Tumor vascularization is necessary to maintain the
increased metabolism of the tumor by faster delivery of nutrients to the cells. Expansion of
blood vessels by the tumor enables metastatic niche formation by tumor-related elements
released into the bloodstream.
Int. J. Mol. Sci. 2022,23, 2261 5 of 24
Furthermore, another extremely important stromal cell subpopulation involved in
tumor progression regulation are mesenchymal stem cells (MSCs). During the EMT process,
the percentage of MSC subpopulation in tumor microenvironment increased. MSCs pro-
duce vimentin, N-cadherin, fibronectin, matrix metalloproteinases, integrins, and smooth
muscle actin, which form a foundation of the tumor extracellular matrix [
57
,
58
]. MSCs
also expressed chemokines involved in the enrollment of immune cells and stromal cells
towards increasing tumor cell mobility and overcoming the venous barrier during neovas-
cularization across EMT. Exosomes excluded from MSC are involved in second tumor niche
formation and the development of chemoresistance. Exosomes include a shortened cancer
repertoire with crucial for disease progression molecules, e.g., proteins and non-coding
regulatory RNAs fraction [
59
]. MSCs, circulating tumor cells, and exosome might be one of
the aims for targeted therapy in metastatic lung cancer [60].
2.3. Immunology of Lung Cancer Tumor
There are two main immune types of cancer: cold tumor and hot tumor. Lack of
tumor-infiltrated immune cells, within the absence of pro-inflammatory cytokines and
chemokines in TME and expression of immunosurveillance factors, are typical for cold
tumors. In contrast, hot tumors are infiltrated by immunosuppressive cells and cytokines,
which activate anergy of T cells and make tumors invisible to the host immune system. In
NSCLC, the “hot type” is associated with high levels of regulatory T cells in TME within
high expression of negative control immune checkpoint inhibitors, PD-L1 and TIM-3, and
is more common for lung adenocarcinomas [
61
]. There is no correlation between immune
type of tumor and the appearance of driver mutations (KRAS and EGFR) [
62
,
63
]. Neverthe-
less, high tumor mutational burden, which is typical for smokers, is connected with better
“visibility” of tumors for immune cells [
64
]. Tumor-infiltrated immune cells may include
cancer-infiltrated cytotoxic T cells (CTLs), memory T cells (CD
45
RO
+
), regulatory T cells
(CD4
+
CD25
+
), tumor-infiltrated macrophages type M1 and/or M2, and dendritic cells. The
infiltration location may appear in the tumor core or its marginal side. The recruitment
of immune cells, and their localization by the tumor side, are regulated by immunosup-
pressive molecules from cancer cells (prostaglandin, histamine, epinephrin, indoleamine
2,3-dioxygenase, arginase, TNF-
α
, TGF-
β
, and IL-10) under metabolomic conditions in
TME (hypoxia). Immune cells present in TME play certain role in tumor–tumor microen-
vironment interaction. Every type of tumor-infiltrated immune cells may promote tumor
progression or provide antitumor activity. During disease development and progression,
cancer cells’ phenotypes become less recognizable for antigen-presenting cells. Involvement
of stromal cells (MSCs and CAFs) into interaction with tumor and immune cells translates
into the silencing of antitumor immunity. Matricellular proteins, such as collagen type I
and III, within CAFs and regulatory T cells form a tumor barrier, which prevents TME
infiltration and metastases propagation. Negative correlation between extensive tumor
invasion and collagen type V/CAFs tumor barrier, formed by flatting CAFs formation and
low concentration of collagen V, was also reported. There were also two types of immune
cellular barrier identified, which correlated with the type of matricellular tumor barrier.
Collagen I/III-CAFs barrier was correlated with a higher presence of CD3
+
and CD8
+
T
cells within high expression of PD-L1 and CTLA-4, whereas collagen V-CAFs barrier was
associated with regulatory T cell presence and exhausted CD8
+
T cell within production
of immunosuppressives factors [
65
]. The immune synapse formation between antigen
presenting cells (APCs) and lymphocyte is a key step for anti-tumor immune response
activation. Tumors use immune checkpoint inhibitory pathways, mediated by cytotoxic
T-lymphocyte-associated protein 4 (CTLA-4), PD-1 (programmed death 1), inducible T-cell
costimulatory (ICOS), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin, and
mucin-domain containing-3 (TIM-3), to prevent T lymphocyte proliferation and activation
of natural killer (NK) cells, which are responsible for cancer cell lysis. Tumor immunology
is a complex issue, with an unquestionable role in the development of cancer treatment.
Immune checkpoint inhibitory (PD-1/PD-L1 and/or CTLA-4 blockade) therapy of NSCLC
Int. J. Mol. Sci. 2022,23, 2261 6 of 24
provides better opportunity for patient response to treatment. Nevertheless, the distinction
between immune therapy and chemo/radiotherapy in patients with NSCLC and frequent
high severity adverse effect during therapy indicates the need to better understand molec-
ular regulations of ICIs in lung cancer. Here, a critical role belongs to pre-clinical study
models. In the presented paper, we describe in brief the main immune cell types and their
function in the tumor–microenvironment interaction. Dendritic cells have the highest pos-
sibility for antigen presentation to helper T cells. DCs take part in the initiation of adoptive
and innate immune response, while the restoration of the adoptive immune response is
beneficial for cancer treatment. Functionally mature dendritic cells’ interaction with NK
cells provides their activation which contributes in tumor cell lysis [
66
68
]. An important
role in the development of immune suppression is held by TME myeloid-derived stem
cells (MDSCs), recruited from bone marrow by IL-1 and IL-6, which are the precursor cells
for granulocytes, macrophages, and dendritic cells. TME MDSCs convert amino acids into
NO and H
2
O
2
, which translates into the inhibition of effector T cells and stimulates the
development of highly hypoxic condition. Myeloid-derived stem cells’ phenotype corre-
lates with disease stage. MDSCs are also involved in the inhibition of T cell proliferation,
exosome recruiting, tumor metastasis, and neovascularization stimulation through cytokine
production under hypoxia condition. MDSCs also stimulate higher expression of PD-L1 on
non-specific response’ cells (DCs or macrophages), hence it is a desirable target for novel
immune therapeutics [
68
72
]. Main subtypes of T lymphocyte corelated with lung cancer
include Th1, regulatory T cell (T
reg
), and cytotoxic CD8-positive T cells. Th1 subpopulation
produce IFN-
γ
and TNF cytokines, while T
reg
lymphocyte is responsible for producing
interleukin 10 (IL-10) and TGF-
β
. These cytokines are involved in immune suppression and
EMT regulation. B cells are common for lung cancer TME, but there are still not enough data
for subpopulation differentiation and its role in cancer immunity [
73
]. Tumor associated
macrophages (TAMs) play a key role in tumor surveillance from immunological control
and EMT regulation. There are two main subtypes of infiltrated TME: pro-inflammatory
M1- subtype and pro-tumorigenic M2-subtype. Under stimulation by stromal CCL2, the
second TAMs subtype produces CCL3, which activates mesenchymal stem cells to produce
exosomes involved in the enhancement of EMT [
74
]. Interaction between tumor cells, CAFs,
and TAMs translates into higher levels of VEGF, matrix metal-proteinases (MMP, mainly
MMP-3 and MMP-10), and selected chemokines involved in EMT [
75
]. Another important
subject connected with tumor metabolism and tumor microenvironment interaction is hy-
poxia. Lung cancer is a highly hypoxic cancer type with two-times lower oxygen percentage
in the tumor side in comparison to normal tissue. Hypoxia limits immune cell infiltration
and conducts tumor surveillance [
76
]. It is also involved in tumor EMT stimulation and
treatment-resistance, mainly in radiotherapy [
77
]. Cuccarese et al. reported TAM infiltra-
tion differences based on cancer molecular type and hypoxia level [
78
]. Hypoxia-inducible
factor 1
α
(HIF-1
α
), but also HIF-2
α
, angiopoietin-2 (Ang-2), fibroblast growth factor (FGF),
and insulin-like growth factors (IGF) are involved in cancer progression (vimentin medi-
ated EMT stimulation) and angiogenesis stimulation (VEGF upregulation) under hypoxia
condition [
79
]. Hypoxia stimulates epithelial-mesenchymal polarization in lung cancer,
promoting its higher aggressiveness and treatment resistance. High levels of TAMs in TME
impacts on the sustaining of tumor hypoxia and exhaustion of TILs. On a metabolomic
level, anaerobic tumor cells complement aerobic TAMs. Association between presence
of driver mutations and hypoxia levels in tumor cells within production of VEGF differs
based on mutational type and localization. A link between factors related to hypoxia and
circulating tumor DNA or miRNA was confirmed and can be potentially applied in clinical
practice [
79
]. HIF-1
α
stimulates tumor neovascularization through involvement of VEGF
family, within upregulation of matrix metalloproteinases secretion and higher activity of
ERK1/2 pathway [
80
]. Inhibition of neovascularization and tumor angiogenesis is one
of the important clinical strategies in treating advanced lung cancer. VEGFR inhibitors,
such as ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, and vandetanib are used in
clinical practice [81].
Int. J. Mol. Sci. 2022,23, 2261 7 of 24
Briefly, characteristics of the main elements of the tumor microenvironment, involved
in tumor progression, angiogenesis, and metastatic promotion, are presented in Figure 2.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 24
metabolomic level, anaerobic tumor cells complement aerobic TAMs. Association be-
tween presence of driver mutations and hypoxia levels in tumor cells within production
of VEGF differs based on mutational type and localization. A link between factors related
to hypoxia and circulating tumor DNA or miRNA was confirmed and can be potentially
applied in clinical practice [79]. HIF-1α stimulates tumor neovascularization through in-
volvement of VEGF family, within upregulation of matrix metalloproteinases secretion
and higher activity of ERK1/2 pathway [80]. Inhibition of neovascularization and tumor
angiogenesis is one of the important clinical strategies in treating advanced lung cancer.
VEGFR inhibitors, such as ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, and
vandetanib are used in clinical practice [81].
Briefly, characteristics of the main elements of the tumor microenvironment, in-
volved in tumor progression, angiogenesis, and metastatic promotion, are presented in
Figure 2.
Figure 2. Characteristics of main elements of the tumor microenvironment, involved in tumor pro-
gression, angiogenesis, and metastatic promotion.
3. Three-Dimensional Preclinical Models for Lung Cancer Study
Herein, mimicking tumor–tumor microenvironment interactions in an in vitro model
required the use of cancer cells and tumor stroma such as scaffolds. This can be supple-
mented by a co-culture of cancer stem cells, immune cells, and normal epithelial cells or
normal fibroblasts. Cancer cells can be obtained from commercial cell lines or derived
from patients. Almost all cell lines were obtained in 70–80-ss, and the dominant histolog-
ical type is primary or metastatic lung adenocarcinoma. The use of cell lines limited ex-
periments from molecular dedifferentiation in comparison to clinical practice. Patient-de-
rived cancer cells can be obtained from surgical excerpts, biopsies, or peripheral blood.
Figure 2.
Characteristics of main elements of the tumor microenvironment, involved in tumor
progression, angiogenesis, and metastatic promotion.
3. Three-Dimensional Preclinical Models for Lung Cancer Study
Herein, mimicking tumor–tumor microenvironment interactions in an
in vitro
model
required the use of cancer cells and tumor stroma such as scaffolds. This can be supple-
mented by a co-culture of cancer stem cells, immune cells, and normal epithelial cells or
normal fibroblasts. Cancer cells can be obtained from commercial cell lines or derived from
patients. Almost all cell lines were obtained in 70–80-ss, and the dominant histological type
is primary or metastatic lung adenocarcinoma. The use of cell lines limited experiments
from molecular dedifferentiation in comparison to clinical practice. Patient-derived cancer
cells can be obtained from surgical excerpts, biopsies, or peripheral blood. Schematic
abbreviation of significant elements in a 3D pre-clinical model is presented in Figure 3.
The type of scaffold and co-cultures used depend on the aim of the study and technical
capabilities. New techniques are used for three-dimensional cell culture studies such as
spheroids, organoids, patient-derived xenograft models, and
in vitro
cancer tissue model
harvested in bioreactor [
82
84
]. At this point, it should be mentioned that nowadays
term “spheroid” is used for 3D cultures derived from commercial cancer cell lines, while
“organoid” is used for patient-derived cancer cells. Based on spheroids and organoids,
cancer-on-chip and 3D printed tumor constructs were developed [
85
]. In brief, for 3D
tumor–microenvironment interaction study, the use of extracellular matrix and immune
cell-co-culture, beside tumor cells, is essential. The type of used scaffold for 3D cell cultur-
ing affects the metabolomic of the entire system: the way tumor cells form spatial structures,
the penetration rate, and the distribution of medium components. For extracellular matrix
imitation, low attachment cell plates, Matrigel, or different modifications of hydrogels con-
Int. J. Mol. Sci. 2022,23, 2261 8 of 24
taining tumor stromal proteins are more commonly used [
86
]. Novel methods for
in vitro
tumor culturing, such as bionic structure, are under investigation. The use of complex
tumor microenvironment 3D models require the development of higher-precision analytical
technical tools and equipment. Several techniques are used for optical detection of tumor
microenvironment metabolic complex, e.g., confocal microscopy, Raman imaging, RPPA,
NMR-based imaging, MSI, and MALDI-based imaging [
87
]. Another method was reported,
namely transparent tissue tomography–3D scanning for analysis of immune checkpoint
inhibitors monoclonal antibody penetration inside the ex vivo tumor model and perme-
ability of the tumor microvascular system. Ex vivo tumor samples were generated from
patient-derived xenograft NSCLC mice [
88
]. Chen et al. used A549 spheroids to investigate
therapeutical effect of hydroxychloroquine on cellular lipidomic. Their matrix-assisted laser
desorption/ionization-mass spectrometry imaging showed differences in type and level of
lipids according to the spheroids’ region location [
89
]. A color-coded tumor tissue model
for lung adenocarcinoma multicellular spheroids was reported by Chan et al. This method
is based on the application of fluorescent proteins, which provides the possibility to moni-
tor interactions between tumors and TME elements within cellular phenotype detection
under confocal microscopy [
41
,
90
,
91
]. An important point in tumor–microenvironment
interaction studies is metabolite production and distribution between tumor cells and
morphological compartments in tumor microenvironments. Tumor spheroids or organoids
are typically harvested after 6–7 days, which is enough for metabolites production studies.
It should be remembered that not every change at the genetic level is reflected at the
protein one. Regarding cell heterogeneity in tumor microenvironment 3D models, the use
of tools enabling the simultaneous transcriptomic analysis of several cell types is desirable.
Single cell sequencing offers a possibility to identify tumor heterogeneity and TME cellular
elements, e.g., creating TME infiltrated immunological cells map [92].
The appropriate selection of individual elements, and at the same time, the complexity
of the culturing system closely correlates with the set goals of the study. In a 3D tumor
microenvironment mimicking model, cancer cells, tumor stromal elements, and tumor
infiltrated immune cells may be used, as well as normal epi- or endothelial cells, normal
fibroblasts, or cancer stem cells. Nevertheless, the type of cells used in co-culture is
predicted by posed hypotheses and research questions. More basial tumor-3D models may
be sufficient for cognitive study, but the use of complex organoid or tumor-on-chip models
is required for preclinical studies. Well-planned and -conducted basic research translates
into a reduction in clinical trials, fewer extra costs, and faster introduction of the drug into
clinical practice. The type and mechanical properties of the scaffold used within tumor
stromal elements, along with the type of cancer cells used and their co-cultures in spheroids
under consideration, significantly impact experiment quality and applicability.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 24
Figure 3. Graphic description of the key elements to mimic tumor microenvironment in vitro.
3.1. The Origin of Used Tumor Cells
The origin of tumor cells commonly includes commercial cell lines or autologous cells
derivate from patient samples collected during surgery (primary tumor model) or from
liquid biopsy (often in metastatic disease stage) (Figure 4). Another practice for obtaining
ex vivo tumor cells is the use of mouse xenografts. Patients’ peripheral blood (liquid bi-
opsy) is predominantly used for obtaining the mononuclear cells and, less often, for CSCs
and exosomes isolation. Liquid biopsy samples also include cancer free DNA, miRNAs,
and tumor-related proteins. This method offers the possibility of more personalized stud-
ies of cancer genetics, but there is a disadvantage in the standardization of this method
between patients [27]. Most of the commercial cell lines were obtained in the 1980s, there-
fore their genetical profile might deviate from those which occur in patients nowadays.
However, the use of commercial cell lines gives a “constant factor” with a characterized
genetical and molecular cellular profile, while patient-derived tumor cells represent a per-
sonalized preclinical model. As already mentioned, study on advanced stage lung cancer
models is critical for application to clinical practice. Herein, cancer cells can be obtained
from patient biopsies and collected for diagnostic research. Both the quality of the sample
and its qualitative and quantitative content depend on the bronchoscopic method [93].
Figure 3. Graphic description of the key elements to mimic tumor microenvironment in vitro.
Int. J. Mol. Sci. 2022,23, 2261 9 of 24
3.1. The Origin of Used Tumor Cells
The origin of tumor cells commonly includes commercial cell lines or autologous cells
derivate from patient samples collected during surgery (primary tumor model) or from
liquid biopsy (often in metastatic disease stage) (Figure 4). Another practice for obtaining ex
vivo tumor cells is the use of mouse xenografts. Patients’ peripheral blood (liquid biopsy)
is predominantly used for obtaining the mononuclear cells and, less often, for CSCs and
exosomes isolation. Liquid biopsy samples also include cancer free DNA, miRNAs, and
tumor-related proteins. This method offers the possibility of more personalized studies of
cancer genetics, but there is a disadvantage in the standardization of this method between
patients [
27
]. Most of the commercial cell lines were obtained in the 1980s, therefore their
genetical profile might deviate from those which occur in patients nowadays. However,
the use of commercial cell lines gives a “constant factor” with a characterized genetical
and molecular cellular profile, while patient-derived tumor cells represent a personalized
preclinical model. As already mentioned, study on advanced stage lung cancer models
is critical for application to clinical practice. Herein, cancer cells can be obtained from
patient biopsies and collected for diagnostic research. Both the quality of the sample and
its qualitative and quantitative content depend on the bronchoscopic method [93].
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 of 24
Figure 4. The origin of cancer cell obtained for 3D lung cancer in vitro model formation.
3.2. Multicellular Spheroids
The most basic 3D culture model for tumor microenvironmental interaction study
consists of multicellular spheroids established on low-attachment well-plates or Matrigel.
Low-attachment scaffolds for multicellular spheroid encapsulation can also be used
[94,95]. Multicellular spheroids include cancer cells within one or two types of normal cell
lines and/or human umbilical vein endothelial cells (HUVECs). One commonly used scaf-
fold mimicking tumor stroma is Matrigel, which includes extracellular matrix proteins
(laminin and collagen type IV), proteoglycans, growth factors, etc., whereas CAFs and/or
normal fibroblasts can be established on agarose or hydrogels. The most popular “source”
of cancer cells in already published research articles are commercial cell lines (e.g., the
commonly used lung adenocarcinoma cell line A549) and fibroblast culture and/or HU-
VECs cells in co-culture [41,54,96,97]. There are some examples of methods for generating
multicellular spheroid within more than one co-culture below. Chan et al. proposed a 3D
model for testing interactions between cancer cells and TME-elements through the use of
four co-culture LUAD cell lines, normal human epithelial cell lines, human fibroblasts,
and autologous cancer stem cells in a ratio 1:1:1:0.2. They also proposed hypoxia condition
use for cell culturing [41]. Jaromi et al. used three dimensional co-culture aggregate con-
sisting of NHLF, HUVECs, and A549/PC9 in ratio 4:3:3 for ABC transporter analysis in
accordance to tumor chemo-resistance [98]. Takahashi et al. reported a center-open disc
method for culturing A549-spheroids with high-density fibroblasts co-culture for better
analysis of tumor–stromal intercellular communication [99]. Kwak et al. developed a tu-
mor-spheroids model established on fibronectin-based Matrigel within co-cultures of hu-
man lung fibroblasts and HUVECs. In this model, cell mobility and structural organiza-
tion within tumor angiogenesis were preserved [100]. Rebelo et al. developed alginate
microencapsulation technic for 3D lung cancer model, established from three types of
cells: non-small cell lung cancer cell line, cancer associated fibroblast, and monocytes de-
rived from peripheral blood. In that culture model, monocytes’ polarization into M2 mac-
rophages was observed within the production of cytokines and metalloproteinases con-
nected with EMT [95]. Cho et al. presented the method for preparing an extracellular ma-
trix through preincubation of a tumor cell line with a pre-adipocytes cell line. Next, it was
decellularized and used for in vitro culturing with tumor spheroids generated from
NSCLC’s cell lines [47,101].
Figure 4. The origin of cancer cell obtained for 3D lung cancer in vitro model formation.
3.2. Multicellular Spheroids
The most basic 3D culture model for tumor microenvironmental interaction study
consists of multicellular spheroids established on low-attachment well-plates or Matrigel.
Low-attachment scaffolds for multicellular spheroid encapsulation can also be used [
94
,
95
].
Multicellular spheroids include cancer cells within one or two types of normal cell lines
and/or human umbilical vein endothelial cells (HUVECs). One commonly used scaffold
mimicking tumor stroma is Matrigel, which includes extracellular matrix proteins (laminin
and collagen type IV), proteoglycans, growth factors, etc., whereas CAFs and/or normal
fibroblasts can be established on agarose or hydrogels. The most popular “source” of cancer
cells in already published research articles are commercial cell lines (e.g., the commonly
used lung adenocarcinoma cell line A549) and fibroblast culture and/or HUVECs cells in
co-culture [
41
,
54
,
96
,
97
]. There are some examples of methods for generating multicellular
spheroid within more than one co-culture below. Chan et al. proposed a 3D model for test-
ing interactions between cancer cells and TME-elements through the use of four co-culture
LUAD cell lines, normal human epithelial cell lines, human fibroblasts, and autologous
Int. J. Mol. Sci. 2022,23, 2261 10 of 24
cancer stem cells in a ratio 1:1:1:0.2. They also proposed hypoxia condition use for cell
culturing [
41
]. Jaromi et al. used three dimensional co-culture aggregate consisting of
NHLF, HUVECs, and A549/PC9 in ratio 4:3:3 for ABC transporter analysis in accordance
to tumor chemo-resistance [
98
]. Takahashi et al. reported a center-open disc method for
culturing A549-spheroids with high-density fibroblasts co-culture for better analysis of
tumor–stromal intercellular communication [
99
]. Kwak et al. developed a tumor-spheroids
model established on fibronectin-based Matrigel within co-cultures of human lung fibrob-
lasts and HUVECs. In this model, cell mobility and structural organization within tumor
angiogenesis were preserved [
100
]. Rebelo et al. developed alginate microencapsulation
technic for 3D lung cancer model, established from three types of cells: non-small cell lung
cancer cell line, cancer associated fibroblast, and monocytes derived from peripheral blood.
In that culture model, monocytes’ polarization into M2 macrophages was observed within
the production of cytokines and metalloproteinases connected with EMT [
95
]. Cho et al.
presented the method for preparing an extracellular matrix through preincubation of a
tumor cell line with a pre-adipocytes cell line. Next, it was decellularized and used for
in vitro culturing with tumor spheroids generated from NSCLC’s cell lines [47,101].
3.3. Hydrogel Based Technics
Several studies point out the impact of hydrogel scaffold composition on the interac-
tion between a tumor and its stromal elements displayed by different level of cytokines
and chemokines production. Herein, hydrogel stiffness and capability to link fibroblast
culture are important parameters for 3D culture formation. Park et al. used A549, HU-
VECs, and human lung fibroblast cell lines to generate spheroids established on hydrogel
with vascular-mimicking channels. This study presents a better
in vitro
model for drug-
dose testing [
102
]. Ferreira et al. developed 3D microspheres from A549, fibroblasts, and
bone marrow derived mesenchymal stem cell on hyaluronic microparticles. The proposed
microparticles are adequate to use for different co-culture combinations and drug test-
ing [
103
]. Li et al. created a silk fibroin and chitosan based scaffold forced tumor spheres
formation [
104
]. Dhamec et al. developed an A549 spheroid with human lung fibrob-
last co-culture in N-isopropylacrylamide-based hydrogel for doxorubicin-drug response
analysis. The proposed model allowed scientists to obtain region with hypoxia [
105
].
Mondrinos et al. proposed a 3D model of lung cancer’s impact on muscle cachexia. For
this study, A549 spheroids established on 3D collagen I hydrogel were used. Interaction
between co-cultures was studied in a microfabricated multichambered device. The use
of a cisplatin-resistance A549 cell line in this model gave a chemoresistance-tumor model
in vitro
[
106
]. Temples et al. showed dependency between integrin density in a hydrogel
system and NK migration. The type and level of produced cytokines and chemokines
related to NK cells differed between each lung cancer cell line used [
107
]. In recent years,
optimalisation of hydrogels for cancer study has been growing. The main points of interest
refer to hydrogels optimalisation for better analysis of metabolomic changes, e.g., hypoxia,
stress relaxation, and hypothermia, but also as a scaffold for ex vivo xenograft. Selecting the
right composition translates into the mechanical properties of the hydrogel and its impact
on formation of 3D cancer model. Blache et al. used polyethylene-glycol hydrogels to form
a 3D platform for simultaneous analysis of transcriptome and secretome [
108
]. Hydrogels
also became a tool for tumor neovascularization study through the mimicking of blood
vessel formation
in vitro
[
104
,
109
118
]. Development of the liquid phase in hydrogels and
tumor-on-chip models provided the possibility for the replacement of the culture fluid
and thus ensuring the flow of cellular metabolites and regulatory molecules [
88
,
116
,
119
].
Bioprinting technology gives a compatible platform to create tumor-on-chip, which in-
cludes tumor stromal elements and is suitable for obtaining multicellular tumor-spheroids
on its surface [
120
]. Veith et al. developed a spatiotemporal apoptosis mapper based
on the tumor-on-chip model for monitoring tumor cell death under chemotherapy or
induced by TILs [
121
]. To imitate metastasis, Ramamoorthy et al. developed a metastatic
tumor-on-dish model, which offers the potential to analyze cell phenotypes within DNA
Int. J. Mol. Sci. 2022,23, 2261 11 of 24
pharmacogenetics sensitivity [
122
]. The possibility of preparing several cultures generated
from patients’ samples based on tumor-on-chip methodology was also reported. This fact
commands the clinical potential of the tumor-on-chip model in personalized medicine,
through next-generation sequencing and a possibility of the precise selection of therapy
(targeted or immunotherapy, mainly by ICIs). The use of the tumor-on-chip model involves
the need to select a suitable analytical model, which could be performed on chip or off chip.
A reliable description of the tumor-on-chip method with its application in clinical practice
was provided by Berzina et al. [
123
]. Another way to obtain cancer cells able to form 3D
structures are ex vivo derivate allografts or xenografts. Further reported are methods for
obtaining decellularized scaffolding for 3D tumor models from mice-xenografts. Strattman
et al. used 3D spheroids on decellularized tissue matrix
in vitro
models in combination
with in silico Boolean model [
124
]. Goliwas et al. proposed a method for the development
of perfusion bioreactor platform to investigate the role of extracellular vesicles in tumor–
TME interaction on a lung cancer model performed from commercial lung cancer cell lines,
fibroblasts, TILs, and exosomes [
125
]. Mishra et al. proposed rat-derivate decellularized
lung as a scaffold for a 3D lung cancer model based on A549 lung adenocarcinoma cell line,
harvested in bioreactor [
126
,
127
]. Patient-derived xenograft models are based on the injec-
tion of cancer cells into mice models for
in vivo
tumor harvesting. This method is expensive
and has some limitations (not all morphological type of lung cancer can be obtained by this
model, mainly NSCLC subtypes) [
128
,
129
]. Padhye et al. proposed the development of
a 3D TME mimicking lung cancer model obtained from previously harvested tumors in
syngeneic mice. Multicellular aggregates consist of all cell types presented in TME. The
use of a murine model provides the opportunity to generate tumors with an activated,
metastatic-process-controlled epithelial–mesenchymal transformation molecular mecha-
nism. Next stage
in vitro
3D culture study allowed identification of miRNA-200 family role
and Src-pathway in regulation of mesenchymal tumor cells involvement in EMT. A laminin
rich matrix was used in that study [
130
]. Bioprinting is a complex issue which becomes
one of the main currents in development of preclinical models. The technical aspects and
application uses of bioprinting methods were presented in review by Augustine et al. [
131
].
A short description of selected cited works is provided in Table 1. Advantages,
disadvantages, and potential applications of presented 3D models are described in Table 2.
Table 1. Short description of selected cited works is provided.
Cellular Components of the Model The Aim of the Model Design Reference
Multicellular
spheroids model
Three-dimensional co-culture: lung
adenocarcinoma cell lines, normal
human epithelial cell line, human
fibroblast and autologous CSC in a
ratio 1:1:1:0.2
Tumor chemo- and
radio-resistance analysis Chan et al. [41]
NCI-H460 and A549 cell lines
established on pre-prepared
decellularized tumor-associated matrix
Role of the fibronectin in
tumor extracellular matrix Cho et al. [47]
Microencapsulation method for
obtaining three-dimensional co-culture:
NCI-H157, lung derived CAFs,
THP-1/peripheral blood derived
monocytes
Prediction of chemo- and
immunotherapy response Rebelo et al. [95]
Three-dimensional co-culture
aggregate: NHLF, HUVECs and
A549/PC9 in ratio 4:3:3
ABC transporter analysis
Tumor chemo-resistance
analysis
Jaromi et al. [98]
Int. J. Mol. Sci. 2022,23, 2261 12 of 24
Table 1. Cont.
Cellular Components of the Model The Aim of the Model Design Reference
Long-term co-culture of A549
spheroids and fibroblasts
Interaction between tumor
and tumor-stromal cells
(fibroblasts)
Takahashi et al. [99]
Three-dimensional co-culture:
MDA-MB-231, MSCs, HLFs and
HUVECs established on collagen-based
Matrigel and fibrin gels, with the
presence of bioengineered blood vessel
Tumor angiogenesis and
blood vessel invasion study Kwak et al. [100]
Hydrogel- based
technics
Spheroidal co-culture: A549, human
lung fibroblasts cell line, HUVECs,
established on hydrogel with
vascular-mimicking channels
Drug-dose testing Park et al. [102]
Three-dimensional microspheres: A549,
fibroblasts, bone marrow derived MSC
established on hyaluronic
microparticles
Drug testing
Adequate method to use for
different co-cultures
Ferreira et al. [103]
Silk fibroin and chitosan-based scaffold
Tumor spheres formation
Neovascularization study Li et al. [104]
Three-dimensional co-culture: A549
and human lung fibroblast established
on N-isopropylacrylamide-based
hydrogel
Doxorubicin-drug response
analysis
Tumor hypoxia study
Dhamec et al. [105]
A549 (cisplatin-resistant) spheroids
established on collagen type I based
hydrogel, cultured in advanced
microfabricated multichambered device
Study of lung cancer impacts
on muscle cachexia
Tumor chemoresistance
study
Tissue anisotropy
examination
Mondrinos et al. [106]
Three-dimensional cell culture: A549 or
H1299 with co-culture of NK-92 cells
established on peptide-functionalized
poly(ethylene glycol)-based hydrogel
Correlation between
integrins density in hydrogel
and NK cells mobility
Temples et al. [107]
MSC activated by secretomes from
different metastatic niches, established
in hydrogel by encapsulation technic
Simultaneous analysis of
transcriptome and secretome
Examination of correlation
between primer and
metastatic tumor
Blache et al. [108]
Tumor-on-chip model
Monitoring tumor-cell death
under chemotherapy or
induced by TILs
Veith et al. [121]
Metastatic tumor-on-dish model
Analysis of DNA phenotype
changes during metastasis Ramamoorthy et al. [122]
Three-dimensional culture: A549 and
HCC827 cultured on See porcine
material In silico Boolean model
Gefitinib sensitivity study
Prediction of targeted
therapy response
Strattman et al. [124]
Int. J. Mol. Sci. 2022,23, 2261 13 of 24
Table 1. Cont.
Cellular Components of the Model The Aim of the Model Design Reference
Three-dimensional cell co-culture:
A549/H358 and IMR90 or A549/H358,
IMR90 and THP-1 cell lines were mixed
with collagen-rich extracellular
matrices and basement membrane, next
cultured in bioreactor
Study on exosomes role in
interaction between tumor
and tumor
microenvironment
Exosomes role in tumor
immunology
Goliwas et al. [125]
A549 cells cultured on decellularized
rat-derived lung in bioreactor
Mimicking tumor growth
in vitro Mishra et al. [126,127]
Multicellular aggregates pre-cultured
in syngeneic mice were established on
laminin rich matrix
Investigation of miRNAs
role in EMT process Padhye et al. [130]
Table 2.
Advantages, disadvantages, and potential applications of presented 3D tumor–tumor
microenvironment in vitro models.
Type of 3D Model Advanteges Disadvanteges
Applicability in Lung
Cancer Diagnostic and
Treatment
Multicellular spheroids
Cancer cells obtained from
commercial cell lines within
the known molecular profile.
Commercial or autologous
cells could be used in
coculture.
Tumor exhibit
heterogeneous features.
Tumor has hypoxia
polarization.
Procedure is less
complicated than for
organoids.
No limitation to access to
patient derived tumor
samples.
Cost effective and less time
consuming (3 days).
Genetic profile may be
different from that in
commercial cell lines,
obtained on 80s.
A constant molecular
profile for each
commercial cell line
allows for reproducible
results but does not
reflect the clinical reality.
Prescreening activity of
potential drug.
Gene regulation and
molecular changes
analysis with gene
editing technique (e.g.,
CRISPR/Cas9).
Study on different
molecular pathways.
The use of commercial
cell lines gives the
possibility for better
statistical analysis and
results’ translation,
because of maintaining
repeatability of cell lines’
genetic and molecular
profile.
Int. J. Mol. Sci. 2022,23, 2261 14 of 24
Table 2. Cont.
Type of 3D Model Advanteges Disadvanteges
Applicability in Lung
Cancer Diagnostic and
Treatment
Organoids
Established from patient
derived tissue samples is a
personalized in vitro model.
Allows obtaining similar to
the clinical “population”
under laboratory conditions.
Has more heterogenic
structure than spheroids
derived from commercial
cell lines.
It could contain other than
tumor cells (e.g., CSC).
Cell source is highly
depending from the
quality of diagnostic
technique
(bronchoscopy) in
patients with
non-operable lung
cancer. The limitation
and a high risk of
low-quality sample.
Tissue samples obtained
during surgery resection,
performed in lung cancer
patients with I-IIIA
disease stage, represent
different than advanced
disease stage’ molecular
profile.
Time consuming (6
days).
Access to patients
derived samples is
needed.
Precise molecular
diagnostic for each
patient.
Potential application in
personalized medicine
prediction in vitro.
Hydrogel-based technics:
Hydrogels can be used as a
scaffold for different
spheroidal culture and
organoids.
Both synthetic and natural
hydrogels have different
stiffness and capacity level,
which transmits into the
metabolomic of the whole
model.
Synthesis and
optimization of hydrogel
scaffolds require
advanced equipment use
and is time consuming.
Analysis of the
correlation between
different types of
tumor-stromal elements,
tumor cells,
tumor-related immune
cells in changeable
proportions.
Bioprinting
(Tumor-on-chip,
tumor-on-dish)
Autologous samples are
used for generation of bionic
tissue/organ samples.
Contains liquid phase.
Could contain blood vessel
mimicking channel.
There is a possibility to take
two types of analysis:
on-chip (limitation of
material loos), and off-chip
(e.g., analysis of fluid
content).
Require high quality
material, including
patient derived samples.
It’s an expensive
technique, which require
advanced equipment.
Interdisciplinary work.
Advanced drug
screening.
Study on disease
development and
progression.
The possibility of
conducting the
examination at the level
of the whole organ.
Bioreactors
A natural decellularized
scaffold is used (e.g., mouse
decellularized lungs).
Metabolites’ changes can be
monitored in real-time
observation.
Long-term culturing could
be provided.
Specialized equipment is
required
A complex procedure.
Required to obtain
potential scaffold from
animal model.
Drug metabolism in
cancer cell and tumor
microenvironments
screening.
Cancer metabolism
study.
Int. J. Mol. Sci. 2022,23, 2261 15 of 24
4. Challenges in Lung Cancer Diagnostic and Treatment
There are two main directions of preclinical study of cancer: cognitive, for better
understanding of the disease, and applied, for investigation of novel diagnostics and
treating tools for clinical practice. However, cognitive and applied study issues follow
directly from clinical challenges in lung cancer diagnostic and treatment. One of the
significant problems is selecting the key predictor and prognostic factors for personalized
clinical practice in every disease stage. The main cause of unsatisfactory results in survival
times, despite the availability of effective therapies, is patients’ diagnoses in advanced
disease stages within local and distal metastasis. Therefore, establishing changes in the
molecular relationship between tumor and its TME during cancer development, and
progression in translating this into the clinical parameters of patients’ conditions, is crucial.
4.1. Radio- and Chemotherapy
Surgery resection and stereotactic radiotherapy are common methods for early stage
lung cancer treatment. Chemotherapy (e.g., paclitaxel, doxorubicin, carboplatin, peme-
trexed, and gemcitabine) is used for adjuvant therapy after surgery resection/radiotherapy
and as first-line therapy in non-resectable, non-targetable patients. A mutual challenge
for chemotherapy, targeted therapy, and immunotherapy is overcoming the frequent oc-
currence of severe side effects during treatment. Chemotherapy is conducted with non-
selective damage on healthy tissues, which contributes to worsening the condition of
patients. According to clinical trial data, adjuvant chemotherapy results in less adverse
effects, and is a more effective response to treatment [
132
]. There is a need to improve
drug dosage in accordance to tissue penetration, overcoming blood barrier and mechanical
barrier of tumor side possibilities via drugs [
133
]. Herein, hydrogel-based 3D tumor–TME
cultures can be used. One of the main directions nowadays is developing tumor-specific
nanoparticles loaded by chemotherapeutical drugs for precision delivery and limitation
of adverse effects [
7
]. The use of the tumor-on-chip model provides an opportunity to
monitor drug penetration through the tumor sphere in real time and for analysis of molec-
ular changes by off-chip methods. Better learning of cancer chemoresistance is highly
needed. There is a possibility that the use of patient-derived material for 3D
in vitro
mod-
els could lead to better understanding of genetic and molecular mechanisms underlying
therapy-resistance.
4.2. Targeted Therapy
Targeted therapy in a group of patients with driver mutations gives them the chance
to extend survival time under treatment. Similar to chemotherapy, there is an important
challenge to overcome the development of drug resistance in cancer. In is way, the next
generations of molecularly targeted drugs are still being developed. The limitation of side
effects’ appearance during treatment in this therapy type is also needed. For example, in a
group of ALK-positive cases, the highest effectiveness according to clinical data was shown
for lorlatinib, but the highest percentage of adverse effects was also observed [
134
]. Crizo-
tinib resistance in ALK-positive metastatic NSCLC patients developed in one year. The, the
next generation of ALK-inhibitors were approved: ceritinib, alectinib, and brigatinib. Based
on the possibility to overcome the brain–blood barrier, alectinib showed higher effective-
ness in brain metastasis treatment. Therapeutic strategies to overcome resistance mutations
include the concomitant use of CDK, c-MET, or mTOR inhibitors [
135
]. In EGFR-positive
NSCLC patients, the selection of used inhibitors is based on mutational profiles. Three
generations of EGFR-inhibitors were developed. For example, afatinib and dacomitinib are
effective in T790M-EGFR mutated patients, but there is a limitation in high doses due to
adverse effects. A phase III randomized clinical trial (NCT02296125) comparing osimertinib
to gefitinib/erlotinib in overall survival time was conducted in non-treated patients with
confirmed mutation in exon 19 deletion or L858R allele. The median overall survival was
longer for patients treated by osimertinib. After 3 years from the beginning of receiving
treatment, 28% in osimertinib subgroup and 9% in gefitinib/erlotinib subgroup were still re-
Int. J. Mol. Sci. 2022,23, 2261 16 of 24
ceiving the drugs. However, scientists observed high grade adverse effects during therapy
in about 40% of patients in all tested groups [136]. Combination therapy with osimertinib
and another third-generation tyrosine kinase inhibitors is under investigation in several
early phase (I/II) clinical studies. Herein, a sufficient challenge is higher range of adverse
effects during combinate therapy. For example, in osimertinib plus savolitinib tests with
patients with mutations in EGFR and MET genes, overall response rate was 30% and high
grade adverse effects occurred in 57% [
137
139
]. Another targeted drug for lung cancer
treatment complies neovascularization inhibitors, within multiple receptor inhibitors (e.g.,
sorafenib), small-molecule tropomyosin receptor kinase inhibitors and serine/threonine
inhibitors or CDK inhibitors [135]. To summarize in brief, the main challenges in targeted
therapy of lung cancer, apart from frequent occurrence of third- and fourth-grade side
effects, is the limited ability of drugs to overcome blood-barrier and brain–blood barrier
within its cancerous tissue penetration properties. The use of three-dimensional tumor–
tumor microenvironment models containing, e.g., blood vessels may be applicable in drug
dose optimization or in examining how to improve pharmacokinetic and pharmacody-
namic properties of the drug. Use of the tumor-on-chip model and bioprinting techniques
based on patient-derived cells, in combination with novel analytical tools (e.g., single cell
sequencing), provides the opportunity to understand the mechanism of drug-resistance
development in every regulatory level, even epigenomic [
19
,
20
,
28
]. In the past years, one of
the main directions in overcoming drugs’ toxicity, is the development of specific matching
nanocarriers loaded by, e.g., doxorubicin or paclitaxel. Herein, 3D tumor–TME
in vitro
models become a cost-effective opportunity to investigate nanocarriers’ penetration rate
and mechanism of action in a heterogeneous tumor and its interaction with TME. An
important factor, in the limitation of side effect frequency during therapy, is personalized
drug-prediction before treatment introduction. The model, potentially applicable into
clinical practice, was proposed by Strattman et al. and is described above [
124
]. Cancer
stem cells in three-dimensional tumoroids play a crucial role in drug-prediction testing and
drug-sensitivity/resistance testing through a gel-free technique or microfluidic platform
according to different research groups [140,141].
4.3. Immunotherapy
The basis of immunotherapy is restoring the efficiency of the host’s immune system
against cancer. It may involve different steps in inducing an immune response. For
the enhancement of cancer antigen presentation and recognition, cancer vaccines were
developed. Vaccines were produced based on use of tumor antigens, MAGA-A3, MUC-
1, TERT, or several tumor-associated antigens (TAA). Nevertheless, major clinical trials
were not continued in phase III. In recent years, the concept of combinational use of
therapeutical vaccines with ICIs has developed. Saavedra et al. reported a CIMAvax-
EGF vaccine which was sufficiency confirmed in advanced NSCLC clinical trials. The
role of the CIMAvax-EGF vaccine is stimulation of EGF-antibodies production [
142
]. The
ongoing clinical trial NCT04298606 is focused on CIMAvax-EGF for the prevention of lung
cancer development [
143
]. Another option for the enhancement of antigen presentation
is the transfusion of sensitized
in vitro
antigen presenting cells, but this method is not
sufficient for advanced disease stage treatment. Action on immune check points on T
cells and specific regulatory receptors on cancer cells indicate the main direction of lung
cancer immunotherapy. The main ICIs used in clinical practice concern the PD-1-PD-
L1 pathway and CTLA-4. Herein, comparable frequency of high-grade adverse effects
during immunotherapy vs. chemotherapy involved advancement in drug dosage and a
therapeutical scheme. Data from clinical trials showed no correlation between lower doses
of nivolumab and lower frequency of adverse effects. There is a clear need to investigate the
correlation between patients’ immune state and regulatory processes developing in TME.
The combination of two ICIs, ipilimumab and nivolumab, showed promising results in
elongating survival times. Again, the long-term use of immunotherapy resulted in serious,
life-threating adverse effects, which might be provoked by over-stimulation of the immune
Int. J. Mol. Sci. 2022,23, 2261 17 of 24
response [
11
,
15
,
144
]. However, better acknowledgement of the regulatory process between
tumor–TME-immune host under treatment is highly needed. Another point is introducing
ICOS, LAG-3, and TIM-3 potential into clinical practice [
145
]. Datar et al. reported no
correlation between expression of LAG-3 and PD-1 inhibitory axis within independent
correlation between expression of PD-1, TIM-3, and LAG-3 and highly proapoptotic T
lymphocyte cell type [146].
In the selection of therapy, it should be taken into account whether the tumor tissue
has inflammatory features. For noninflammatory types, innate or adoptive immunotherapy,
CAR-T, and targeted therapies could be used. On the other hand, for inflammatory types,
anti-PD-1/PD-L1 and/or anti-CTLA4, active innate (IDO inhibitors and cytokines), or
active adoptive immune therapy could be used. Herein, the investigation of high precision
predictive factors for clinical practice use by immunoprofiling is needed [
147
]. The use
of autologous cells in three-dimensional tumor models based on liquid-phase tools and
various extracellular-matrix compositions is one potential approach to discovering prog-
nostic and predictive factors for immunotherapy clinical use. Investigation of correlation
mechanisms between tumors, immune cells, and tumor stromal elements and their trans-
lation into organism level has the potential to overcome immunotherapy gaps in clinical
settings [
148
150
]. The potential tool to develop high quality analytical platforms may
improve through the use of advanced 3D tumor–TME preclinical models.
5. Conclusions
We presented critical points for tumor–tumor microenvironmental interaction within
brief characteristics of
in vitro
strategies for obtaining 3D tumor microenvironmental mod-
els. The way to choose better pre-clinical models is predicted by the aim of the planned
study and is limited by the availability for patient-derived material, technical background,
and funding. In the field of cognitive study on lung cancer, future perspectives in tumor
microenvironmental interaction studies may include:
Investigation of CSCs role in second niche formation;
Analysis of TILs differentiation mechanism in TME;
Analysis of cells’ phenotype polarization in accordance to culturing conditions;
Exploring knowledge on tumor–TME interaction;
Investigation of new regulatory and tumor developmental mechanisms.
Future perspectives for applied studies on tumor–TME interaction is predicted by
clinical trial data. The main hallmarks of lung cancer diagnostics include:
Prognostic factors from liquid biopsy identification (e.g., potential use of exosomes,
cfDNA, and CSC);
Identification and validation of elements derived from tumor side.
The challenges in lung cancer therapeutical strategies concern:
Improvement in pharmacokinetic and pharmacodynamic drugs’ properties;
Overcoming drug resistance;
Discovering combined therapies;
Discovering new regulatory mechanisms in tumor–TME relationships;
Reduction in adverse effects frequency and grade;
Drug dosage optimalisation;
Higher specificity in drug delivery (e.g., specifically matched nanocarriers for drug delivery).
Advantages in bioprinting technologies and a large variety of analytical methods make it
possible to provide highly specific and personalized pre-clinical studies, with faster applicability
into clinical use and no necessity to involve costly and less effective animal models.
Author Contributions:
I.W. conceptualization, writing, original draft preparation, K.W.-K.; writing,
review and editing, P.K. review, editing, and J.M. review, editing. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Int. J. Mol. Sci. 2022,23, 2261 18 of 24
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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