Having examined a multiplicity of existing and putative immunotherapeutic
targets for the control of cancers we now present several observations which
may act as a basis for extension. Some of the observations here are more
detailed upon what we discussed earlier and others are anticipatory of possible
extensions[1].
The first two, the tumor micro environment and tumor
associated macrophages are we believe critical factors to be considered while
examining immunotherapy. They build upon one another creating a powerful
self-sustaining stronghold for cancer clusters.
Note that the tumor stroma plays an equally important role.
As Bremnes et al have noted[2]:
Maintenance of both normal epithelial tissues and their
malignant counterparts is supported by the host tissue stroma. The tumor
stroma mainly consists of the basement membrane, fibroblasts, extracellular
matrix, immune cells, and vasculature. Although most host cells in the
stroma possess certain tumor-suppressing abilities, the stroma will change
during malignancy and eventually promote growth, invasion, and metastasis.
Stromal changes at the invasion front include the appearance of
carcinoma-associated fibroblasts (CAFs). CAFs constitute a major portion of the
reactive tumor stroma and play a crucial role in tumor progression. The main
precursors of CAFs are normal fibroblasts, and the trans-differentiation of
fibroblasts to CAFs is driven to a great extent by cancer derived cytokines
such as transforming growth factor. During recent years, the crosstalk between
the cancer cells and the tumor stroma, highly responsible for the progression
of tumors and their metastasis, has been increasingly unveiled.
A better understanding of the host stroma contribution to
cancer progression will increase our knowledge about the growth promoting
signaling pathways and hopefully lead to novel therapeutic interventions
targeting the tumor stroma. This review reports novel data on the essential
crosstalk between cancer cells and cells of the tumor stroma, with an emphasis
on the role played by CAFs. Furthermore, it presents recent literature on
relevant tumor stroma- and CAF-related research in nonsmall cell lung cancer. …
The tumor stroma basically consists of
(1) the nonmalignant cells of the tumor such as CAFs,
specialized mesenchymal cell types distinctive to each tissue environment,
innate and adaptive immune cells, and vasculature with endothelial cells and
pericytes and
(2) the extracellular matrix (ECM) consisting of
structural proteins (collagen and elastin), specialized proteins (fibrilin,
fibronectin, and elastin), and proteoglycans[3]
Angiogenesis is central for cancer cell growth and
survival and has hitherto been the most successful among stromal targets in
anticancer therapy. Initiation of angiogenesis requires matrix
metalloproteinase (MMP) induction leading to degradation of the basement
membrane, sprouting of endothelial cells, and regulation of pericyte attachment.
However, CAFs play an important role in synchronizing these events through the
expression of numerous ECM molecules and growth factors, including transforming
growth factor (TGF)-β,
vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF)
2.
We can also call this in certain circumstances the tumor
microenvironment, TME. Historically immune escape was a significant factor. As
Prendergast noted[4]:
Immune escape was not widely recognized among cancer
geneticists or molecular cell biologists as a fundamental trait of cancer until
recently. In the late 1960s, studies of immune deficient nude mice, newly
developed at the time, argued that they had no increased susceptibility to
spontaneous cancers. An influential interpretation of these findings was that
immunity was not a critical restraint to tumorigenesis in mammals.
However, this interpretation was flawed by the lack of
knowledge that nude mice retain natural killer cells (NK cells), which have
potent antitumor activity. Studies of mutated oncogenes discovered in the late
1970s and 1980s tended to reinforce the notion that immunity was not critical
for tumorigenesis, based on demonstrations that malignant cells could be
created from normal cells in vitro.
Through the 1990s, the perspective of many cancer
geneticists and cancer cell biologists was that unmutated genes had relatively
limited roles in cancer pathophysiology, and it was apparent that overt immune
regulatory genes were not mutated in cancer. Later, studies in transgenic mouse
models and sound clinical documentation of the reality of dormant cancers
forced a greater appreciation of how inflammation and immunity contributed to
tumorigenesis.
For example, tumors arising in patients who had received
a transplant from a donor who years earlier had been cured of cancer were found
to be derived from the donor, arguing that occult tumor cells from the
transplant could be immunologically managed for long periods as dormant disease
until they were moved to an immunosuppressed organ recipient (here, it should
be emphasized that the clinical ‘cure’ achieved in the donor was simply a
reduction of the disease to a dormant occult state). In mice, tactics to
genetically ablate T-cell function dramatically increased the incidence of
spontaneous solid tumors. These findings demonstrated that adaptive immunity performs
an essential tumor suppressor function in mammals.
Furthermore, they implied that immune escape is essential
for the formation of a tumor. Recent experimental findings directly corroborate
the notion that tumor cells can exist in an occult state of immune equilibrium
for long periods. In a classical model of chemical carcinogenesis, Schreiber
and Smyth and colleagues showed that immune depletion will reveal tumors in
mice that will remain tumor free after low doses of carcinogen that are
insufficient to trigger tumor formation during the host's lifespan. Evidence of
transformed but dormant tumor cells was obtained in animals along with a
demonstration that such cells are more immunogenic than those present in frank
tumors.
Thus, along with other cell-extrinsic traits of cancer,
immune escape is an essential trait for the development of progressive disease,
acting as a biological modifier to dictate the outcome of an oncogenically
initiated lesion that may otherwise be eliminated or be present in an extended
occult state of immune equilibrium corresponding to dormancy.
Here, the definition of a cancer modifier is broadly
defined in pathological terms as a gene that is phenotypically silent unless
evaluated in the context of cancer. By this definition, many genes influencing
cell-extrinsic traits of cancer—angiogenesis, invasion, metastasis and immune
escape—are understood as modifiers that dominate tumor outcomes.
In short, while mutations in oncogenes and tumor
suppressors start cancer, modifiers and the microenvironment which act later
may dictate their clinical relevance. Learning how immune escape evolves during
the integrated processes of oncogenesis and immunoediting may therefore yield
more powerful insights into cancer pathophysiology and therapy than achieved to
date.
Our focus is based upon the papers by Marin-Acevedo et al
who provided an excellent systematic survey of various immunotherapeutic
approaches[5].
The most well know has been the PD-1 and CTLA4 antibody approaches facilitating
the immune systems in its attack on various cancers. The second dimension is
the CAR-T cell approach and the CD19 targets on hematopoietic cancers. In
previous papers we also examine the use of CIK, cytokine induced killer cells
using the innate system and NK cells[6].
However, it is well known that there are a multiplicity of
other well-known ligands and the like as well as various innate system methods
and also other cell lines which can attack various malignancies. On top of
these there most likely an equal number or more that we have yet to discover.
Our objective herein is to follow Marin-Acevedo et al and to use their outline
and fill it in with other details while allowing for putative expansion in new
markers and ligands.
We have mentioned the TME previously and in some detail in
the introduction. However, it is important to understand that attempting to use
the immune system one must deal with the totality of a tumors defenses and key
amongst them is the TME. As Murgaski et al have noted[7]:
It is becoming increasingly apparent that our immune
system is capable of fighting cancer. Understanding the interplay between our
immune system and cancer has led to the development of new treatments that can
prolong survival in once-thought terminal patients. The success that immune
checkpoint inhibitors (ICIs) have had in the clinic has sparked renewed
interest and investment in the tumour immunology field. However, durable
responses to immunotherapy are only seen in a minority of patients.
A common trait among many treatment responsive patients
is a high neoantigen load; a characteristic which often correlates with a
strong adaptive immune response against the tumour. This response is required
for the ICIs to release the brakes that the tumour places on the immune system.
On the other hand, patients who do present a high
neoantigen load may not respond to ICIs due to an immunosuppressive tumour
microenvironment (TME). In these patients, anti-tumour immune responses are
shut down by immunosuppressive cells such as tumour-associated macrophages
(TAMs) and myeloid-derived suppressor cells (MDSCs). Interactions between these
suppressive cells and effector T cells can lead to T-cell exhaustion, a state
of T-cell dysfunction seen during chronic inflammation.
While ICIs can rescue some T cells from these
interactions, suppression of the TME might still be too strong for T cells to
fully overcome this obstacle, resulting in continued tumour progression. Therefore,
it is imperative to improve the adaptive immune response against the tumour
while simultaneously redirecting the TME toward a more immuno-permissive state.
In this light, harnessing the potential of dendritic cells (DCs) that reside
within tumours is one avenue of research that could yield positive clinical
results for cancer patients in the near future.
It is well established that DCs have the ability to link
both the innate and adaptive immune systems and to initiate immune responses.
In the age of immunotherapy, this capacity to generate adaptive immune
responses is considered to be imperative. DCs can activate T-cell responses…
with great efficacy due to their high expression of co-stimulatory molecules
and specific T-cell adhesion molecules. However, DCs are also capable of
shutting down immune responses by expressing high levels of co-inhibitory
molecules. Therefore, understanding and exploiting mechanisms relating to the
function of tumour-associated DCs (TADCs) can lead to the development of powerful
tools to fight cancer.
Macrophages search out and target cells to be cleaned but by
the immune system, most of the time. However, the tumor associated macrophages
can act in a pro tumor manner actually enhancing tumor growth and activating
metastatic behavior. As Huang et al note[8]:
Tumor associated macrophages (TAMs) play an important
role in tumorigenesis and progression. TAMs generate an inflammatory
environment to trigger or facilitate tumor initiation, promote tumor cell
invasion and metastasis, stimulate angiogenesis and suppress antitumor
immunity. High density of TAMs was correlated with the poor prognosis of a wide
range of tumors such as lung, hepatocellular, colorectal, breast, prostate,
ovarian and thyroid cancers.
TAMs produced growth factors (e.g. VEGF, EGF, HGF and
bFGF) and chemokines (e.g. CXCL12 and IL8) to mediate their oncogenesis
function. On the other hand, cancer cells recruit TAMs by releasing colony
stimulating factor (CSF1), granulocyte–monocyte (GM-CSF), transforming growth
factor (TGF) or chemokines (e.g. CCL2)
In several thyroid excisions one can see the follicular
and/or papillary cells but at the same time if one looks there may be large
collections of macrophages. If that were to be the case then one may expect
that the lesion has metastasized.
Noy and Pollard have noted[9]:
Macrophages in the Primary Tumor: Cancer Initiation
Tumors acquire mutations in oncogenes or tumor-suppressor genes that permit
them to progress to malignancy. Although most cancer research has focused upon
these changes and most therapeutics are directed against these tumor cells, it
is now apparent that the nonmalignant cells in the microenvironment evolve
along with the tumor and provide essential support for their malignant
phenotype.
In fact both the systemic and local environment play a
tumor-initiating role through the generation of a persistent inflammatory
responses to a variety of stimuli. For example, obesity is associated with
increased risk of many but not all cancers and is characterized by an enhanced
systemic inflammatory response and locally, for example in the breast, to an
increased number of inflammatory crown-like structures consisting of macrophage
and adipocytes whose number strongly correlates with breast cancer risk.
Similarly persistent inflammation referred to as
‘‘smoldering inflammation’’ caused by chronic infection with viruses such as
Hepatitis B virus in liver, bacteria like Helicobacter pylori in the stomach,
or due to continuous exposure to irritants such as asbestos in the lung is
casually associated with cancer initiation.
Furthermore, inflammatory conditions such as Crohn’s
disease dramatically increase the risk of colorectal cancer. Inflammation
always has a substantial macrophage involvement through their production of
molecules such as interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), and
interferon-g (IFN-g).
To support this correlative data between
macrophage-mediated inflammation and cancer induction, … found that genetic
ablation of the anti-inflammatory transcription factor Stat3 in macrophages
results in a chronic inflammatory response in the colon that is sufficient to
induce invasive adenocarcinoma. In addition, loss of the anti-inflammatory
cytokine IL-10 that acts through STAT3 enhances carcinogen-induced
tumorigenesis in the intestine.
Mechanistically, this inflammation can cause tumor
initiation by creating a mutagenic microenvironment either directly through
free radical generation or indirectly via alterations in the microbiome and
barrier functions that allow access of genotoxic bacteria to the epithelial
cells.
In fact, there are also metastasis associated macrophages,
MAMs, which are a special class of TAM, in that they assist in the metastatic
process. The TAMs actually suppress and block T cell action as well as NK and
NKT cell actions. The TAMs create a protective buffer for the growth of the
tumor and in a sense add an additional element to the tumor micro environment.
In a strange sense it is the immune system itself which assists in the growth
of the malignancy. The authors continue:
In addition to these MHC molecules, macrophages express
the ligands of the inhibitory receptors programmed cell death protein 1 (PD-1)
and cytotoxic T-lymphocyte antigen 4 (CTLA-4). These inhibitory ligands are
normally upregulated in activated immune effector cells such as T cells, B
cells, and NK T cells as part of a safety mechanism that controls the intensity
of the immune response and as part of inflammation resolution. Activation of
PD-1 and CTLA-4 by their ligands (PD-L1, PD-L2, and B7-1 [D80], B7-1 [CD86],
respectively) directly inhibits TCR and BCR signaling.
This activation also inhibits T cell cytotoxic function,
regulates their cell cycle, and inhibits their activation as CTLA4 competes
with CD28 (costimulatory) binding. PD-L1 and PD-L2 are differentially
expressed, with PD-L1 constitutively expressed by immune cells including T
cells, B cells, macrophages, DCs, nonhematopoietic cells, and cancer cells.
In contrast, PD-L2 expression is limited to antigen-
presenting cells (APCs). Its expression is induced in monocytes and macrophages
by CSF1, IL-4, and INF-g. Both PD-L1 and L2 are regulated in TAMs and
myeloid-derived suppressor cells.
Recently, …showed that MDSCs and TAMs in hypoxic tumor
regions upregulate the expression of PD-L1 as a consequence of HIF-1a signaling
(Noman et al., 2014). Hypoxia acting via hypoxia inducible factor 1- a (HIF-1a)
also induces T cell suppression by TAMS although the mechanism is unknow. It
has also been shown that monocytes from blood of glioblastoma patients express
higher amounts of PD-L1 compared to healthy donors and that
glioblastoma-cell-conditioned medium can upregulate PD-L1 expression in
monocytes from healthy donors.
Similarly, monocytes from patients with hepatocellular
carcinoma express PD-L1 that contributes to human tumor xenograft growth in
vivo, while the blocking of PD-L1 reverses this effect.
Thus, in a strange way the action of the macrophages sets up
the PD-1 type of blockade we then try to work around. Perhaps as some author
suggest we should also target the TAMs and MAMs.
[2] Bremnes
et al, The Role of Tumor Stroma in Cancer Progression and Prognosis, Journal of
Thoracic Oncology • Volume 6, Number 1, January 2011
[3]
See https://www.researchgate.net/publication/333704252_EMT_lncRNA_TGF_SMAD_and_Cancers
and https://www.researchgate.net/publication/330222973_EMT_and_Cancers
also see https://www.researchgate.net/publication/325046881_PCa_mir34_p53_MET_and_Methylation
[4] Prendergast,
Immune escape as a fundamental trait of cancer: focus on IDO, Oncogene volume
27, pages 3889–3900 (26 June 2008)
[5] Marin-Acevedo
et al, Cancer immunotherapy beyond immune checkpoint inhibitors, Journal of
Hematology & Oncology (2018) 11:8
Marin-Acevedo et al, Next generation of immune
checkpoint therapy in cancer: new developments and challenges, Journal of
Hematology & Oncology (2018) 11:39
[7] Murgaski
et al, Unleashing Tumour-Dendritic Cells to Fight Cancer by Tackling Their
Three A’s: Abundance, Activation and Antigen-Delivery, Cancers 2019, 11, 670
[8] Huang
et al, Follicular thyroid carcinoma but not adenoma recruits tumor associated
macrophages by releasing CCL15, BMC Cancer 2016
Huang et al, Vascular normalizing doses of
antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment
and enhance immunotherapy, PNAS, October 23, 2012, vol. 109, no. 43,
17561–17566
[9] Noy
and Pollard, Tumor Associated Macrophages: From Mechanisms to Therapy,
Immunity, 2014
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