Resistance is an innate or acquired response to external stimuli aimed to decrease adverse effect and promote individual survival. Resistance is an important survival mechanism, and allows an organism to adapt and evolve against evolutionary pressures. However, in the field of disease and pathology, the survival of causative agents of infection and cancer can be promoted by resistance. Therefore, resistance functions as an opponent in the case of therapeutics, because it allows the target to escape and evolve against the treatment. A primary resistance towards external factors is derived from germ-line phenotypic expression, and is termed as innate. Whereas, a secondary resistance develops during the course of exposure and adaptation towards the external factors, and therefore is termed as adaptive. These concepts are applicable in understanding the mechanisms by which tumor cells exhibit resistance against immune responses. Tumor cells exhibit both innate and adaptive mechanisms through which they can overcome the host immune response and cumulate in cancer establishment. A brief diversity of mechanisms is discussed below through which tumor cells can impede a myriad regulations imposed by the immune system during cancer initiation and development.
Innate/primary mechanisms of tumors to evade immune response
Primary resistance has a characteristic feature of immediate response against external pressure. These innate mechanisms are constitutively present and exhibit faster kinetics of resistance by tumors against immune responses. Following are some well studied mechanisms exhibited by individual tumor cells, or mechanisms that arise from a complex interaction between tumor-cells, stromal-cells, extracellular matrix (ECM) and secreted factors in the tumor-microenvironment.
Mechanisms exhibited by individual tumor cells: Tumor cells are generated via accumulation of random mutations in the presence of various stimuli. Many of these mutations are silent and do not prove to benefit tumor cells in terms of survival, however, certain mutations in immunomodulatory proteins can aid in being detected or killed by immune cells.
To avoid detection by immune system, some tumor cells mimic a normal cell phenotype. For example, tumor cells downregulate secretion of danger-associated molecular patterns (DAMP) and stress signals to avoid activation of local immune cells. In some cases, tumor cells have also been observed to maintain an optimal level of HLA-1 expression to escape being detected by NK-cell. When detected by T-cells or NK-cells, tumor cells can avoid extracellular cell-death pathways like FAS-mediated apoptosis due to a downregulation or mutations in surface receptors related to these pathways.
Another way of escaping tumor cytolysis is by expression of regulatory checkpoint surface molecules by tumor cells to inactivate the immune effector function. For example, tumor cells were found to express do-not-eat me signals (CD24, CD47) that suppress macrophage mediated phagocytosis by activation of signal-regulatory protein alpha (SIRP-a). Moreover, tumor cells express checkpoint ligands PDL1 and CTLA4 that inhibit T-cell mediated cell death. Here PDL1 is a negative checkpoint inhibitor of T-cell activity that binds to PD-1 of CD28 family of receptors on T-cell surface. PDL1 expression on tumor cells engages T-cells targeting tumor associated antigens and impairs the effector response by reducing their proliferative function. Inversely, PDL1 binding to PD-1 receptors on regulatory T cells reduces the frequency of Treg apoptosis, thus contributing in promoting anti-inflammatory responses. Clinical studies have shown an improvement in patient survival when monoclonal antibodies like durvalumab, pembrolizumab, atezolizumab and avelumab, targeting the PD1-PDL1 axis were used as therapeutics.
In the context of oncogenic virus induced cancers there is more diversity in escape mechanisms where these viruses either exploit existing pathways or the activity of viral proteins for immune-suppression. For example, Epstein-Barr virus (EBV) encodes for IL-10 homolog that prevents T-cell activity and promotes tumor growth. EBV also inhibits expression of adhesion molecules (like LFA-3 and ICAM-1), thus preventing adhesion of lymphocytes to infected cells and promotes tumor escape. Whereas in human papillomavirus (HPV) related cervical cancers, active immune evasion of HPV is mediated intracellularly by altered gene expression (IFN and cGAS-STING) and disturbed protein functions (TLRs), and extracellularly by interfering with immune cell networks from antigen presenting cells to effector T cells.
Mechanisms exhibited by tumor-microenvironment (tumor cells, stromal cells, ECM, soluble factors): The cancer-immune cycle develops in the very surrounding of a local microenvironment that consists of non-tumor and non-immune players. Such a tumor-microenvironment is made up of stromal cells contributing in the tumor volume, epithelial cells regulating vasculature, the extracellular matrix making up the structure and scaffold of solid tumors and a plethora of soluble factors containing but not limited to cytokines, hormones, extracellular vesicles, and growth factors. Therefore, there is an interplay between the tumor-microenvironment and tumor cells that gives rise to various mechanisms discussed below that can promote tumor immune escape.
Tumor cells exploit metabolic pathways to create a microenvironment that is antagonistic to effector immune cells. For example, it has been shown that tumor cells participate in Warburg effect, where they exhibit an increased uptake for extracellular glucose. This leads to glucose deprivation in the microenvironment where resident immune cells fail to compete and convert to a resting and non-effector phenotype. Moreover, anaerobic glycolysis of glucose produces a large amount of lactate that is secreted in the extracellular area by tumor cells. Such an acidic environment proves to be antagonistic for the function of effector T-cells, but not regulatory T-cells (Tregs) and myeloid derived suppressor cells (MDSC).
Chronic inflammation in the tumor microenvironment induces constitutive activation and presence of reactive species released by macrophages and neutrophils to induce tumor cell death. However, tumor cells can inhibit or scavenge these extracellular reactive species by secretion of catalase and superoxide dismutase to maintain tolerogenic environment. Simultaneously, these reactive species contribute in providing genomic stress that promotes accumulation of mutations in surviving tumor cells, resulting in tumor evolution. In another set of circumstances, tumor cells can promote wound healing phenotype of myeloid derived cells and render them anti-inflammatory via secretion of various angiogenic stimuli like VEGF. In the context of secreted signals, tumor cells produce anti-inflammatory cytokines like IL-10 and TGF-b to promote proliferation of regulatory immune cells like MDSC and Tregs in the microenvironment. Additionally, this promotes the differentiation of naive immune cells to develop and exert a regulatory phenotype. Eg: PBMC derived monocyte differentiate to MDSC in presence of IL-4 or IL-10 secreted by tumors.
Recent elucidation of extracellular vesicle mediated cell-to-cell communication has shed some light on how tumor cells can exploit this to educate other cells present in the microenvironment. Various studies have shown that tumor cell-derived vesicles contain micro-RNA and regulatory proteins that change the behavior and function of the recipient cell. For example, tumor cells secrete vesicles containing surface-PDL1 that prevents the proliferation of effector T cells. Moreover, these tumor-derived vesicles have been found to secrete pro-tumoral non-coding RNA fragments that can educate stromal cells to change their metabolic state or to secrete anti-inflammatory factors in the microenvironment.
Adaptive/secondary mechanisms of tumor to escape immune response
As we have learnt in previous chapters, cancers evolve through repetitive process of clonal expansion, genetic diversification and clonal selection amidst a dynamic microenvironment. Immune responses can eradicate susceptible clones but it can also unintentionally provide a selective pressure for the expansion of resistant variants. Accordingly, such secondary resistance against anti-tumor immunity is generated via somatic changes that evolve during immune response. This is a well studied example of arms-race between the tumor and immune system, where the dynamic cancer-immune cycle simultaneously evolves for both a better anti-tumor response and novel immune escape mechanisms for tumor survival. These adaptive mechanisms are delayed in terms of onset due to the time required for evolution but are nevertheless long-term due to selection of the most resistant mechanisms in time.
Clonal selection in cancer: The early immune responses against tumor initiation and progression lead to a phenomena called ‘Immunoediting’ where inherently immunogenic tumor cells are eliminated. Later, less-immunogenic tumor cells survive and rec-colonize the tumor. Such phenomena can result from point mutations that may not be immunogenic (Eg: mutations in RAS proteins) but can be tumorigenic. Moreover, complete loss of tumor suppressor proteins like TP53, also fails to generate tumor associated antigens. Such tumor cells accumulate mutations due to existing sub-optimal DNA damage repair system, which allows them to evolve for non-immunogenic antigens. Tumor cells can also participate in ‘antigen masking’, where antigens are hidden from the immune system via post-translational modifications like glycosylation or sialyation.
Tolerization of tumor antigens in the absence of co-stimulation: Failure of tumor cells and antigen presenting cells to express right levels of MHC-1/2 and co-stimulatory signals can impede the activation T-cell specific responses. Therefore, even in the presence of proper tumor associated or tumor derived antigens, the respective reactive T-cells may not be stimulated for effector functions. Here the tolerogenic tumor microenvironment plays an important role in regulating the activation state of primary antigen presenting cells like dendritic cells. Additionally, anti-inflammatory signals produced by tumor resident myeloid cells prevent the clonal expansion of the epitope-specific T-cells and thus aid in immune evasion.
- Resistance: the ability of an organism to resist harmful influences (such as disease, toxic agents, or infection)
- innate: applies to qualities or characteristics that are part of one’s inner essential nature
- adaptive: qualities or characteristics that arise as a result of adaptation and evolution to external influences
- evolutionary pressure: The effect of external influences on an organism in course of time. Any change in the environment that encourages particular mutations to succeed. For example, antibiotic use kills susceptible bacteria and allows microorganisms with resistant genes to survive and proliferate.
- extracellular matrix (ECM): Non-living material secreted by cells that fills spaces between the cells in a tissue, protecting them and helping to hold them together. The extracellular matrix may be semifluid or rigidly solid and hard as in bone. It is composed mainly of protein and includes collagens, elastin, reticulin, glycoproteins, proteoglycans, fibronectin, laminins and osteopontin.
- danger-associated molecular patterns (DAMPS): Molecular signals that activate immune response by acting as an indicator of unhealthy cells in the local environment.
- HLA-1: The class 1 of major histocompatibility antigens in humans, involved in presentation of intracellular antigens.
- HLA-2: The class 2 of major histocompatibility antigens in humans, involved in presentation of extracellular derived antigens.
- Fas-mediated apoptosis: A pathway for programmed-cell death or apoptosis mediated from extracellular signals, in this case via FAS-ligand.
- Do-not eat me signal (CD24, CD47): Surface proteins expressed on cells that inhibit macrophage mediated phagocytosis.
- Checkpoint regulators (CTLA4, PDL1): Surface proteins expressed by target cells to inhibit T-cell mediated cytotoxic effects.
- Epstein-Barr virus (EBV): DNA virus of herpes family that infects B cells and epithelial cells, and is a causative agent of various malignancies.
- Human papillomavirus (HPV): DNA virus from the papillomaviridae family, and a major causative agent of cervical cancers.
- Interferon (IFN): Cytokines that are secreted upon viral infection, and ‘interfere’ in viral replication in neighboring cells via inducing a diverse immune responses.
- cell adhesion molecules (LFA-3, ICAM-1): Surface proteins that are involved in binding with other cells or with the extracellular matrix (ECM).
- tumor-microenvironment: The tumor microenvironment (TME) is the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix (ECM).
- regulatory T-cells (Tregs): These suppressor T cells modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease via downregulation of induction and proliferation of effector T cells.
- myeloid derived suppressor cells (MDSCs): These are a heterogenous group of immune cells of myeloid origin from bone marrow stem cells and possess strong immunosuppressive activities rather than immunostimulatory properties.
- chronic inflammation: Chronic inflammation is the result of release of pro-inflammatory cytokines from immune-related cells and the constitutive activation of the innate immune system.
- vascular endothelial growth factor (VEGF): It is a signal protein that stimulates the formation of blood vessels.
- extracellular vesicles: Extracellular vesicles (EVs) are non-replicating lipid bilayer particles that are naturally released from a cell. Vesicles of endogenous origins are termed exosomes, while membrane originating vesicles are called microvesicles.
- microRNA: It is a small non-coding RNA molecule (~ 22 nucleotides), that functions in RNA silencing and post-transcriptional regulation of gene expression.
- immunoediting: It describes the relation between the tumor cells and the immune system and functions as a dynamic process that consists of immunosurveillance and tumor progression. It is made up of three phases: elimination of immunogenic tumor cells, equilibrium between immune surveillance and tumor eradication, and escape of low-immunogenic tumor cells.
- antigen masking: The phenomena where antigens are hidden from the immune system and made less-immunogenic via post-translational modifications like glycosylation or sialiation.
- tolerance: Immune tolerance is a state of unresponsiveness of the immune system to substances or tissue that have the capacity to elicit an immune response in a given organism.