Abstract
Success of cancer immunotherapy (CIT) is intricately influenced by the tumor microenvironment (TME), a complex ecosystem that encompasses immune cells, stromal elements, and extracellular components. Despite the clinical breakthroughs of immune-checkpoint inhibitors (ICIs), adoptive cell therapies, cancer vaccines, and other immunotherapeutic interventions, many patients fail to respond and eventually die. Emerging evidence points to cancer stem cells (CSCs) as critical drivers of immune evasion, therapy-resistance, and tumor relapse. CSCs modulate the TME by secreting immune-suppressive factors, recruiting regulatory immune cells, and inducing phenotype-switching of anti-tumor TME subsets, thereby creating a protective niche that hinders immune surveillance. Conversely, the TME protects CSCs through hypoxia, altered metabolism, and immuno-suppressive cell populations. This bi-directional crosstalk supports tumor progression and provides resistance to immunotherapeutic strategies mainly by: (i) escaping immune-recognition and inhibiting active T cells via high immune-checkpoint molecule expression, (ii) creating immunosuppressive pro-tumor environment, and (iii) evading immune-mediated apoptosis of CSCs along with therapy-induced enrichment of their pool. Targeting CSCs in concert with reprogramming the TME via CSC-directed agents, metabolic modulators, or combinatorial immunotherapies, therefore, offers a promising avenue to overcome immunotherapy-resistance and achieve durable clinical responses. This review discusses the deeper mechanistic understanding of CSC-TME interactions, in light of designing next-generation immunotherapies with broader efficacy across diverse tumor types.
Keywords
Introduction
Tumor microenvironment (TME) refers to the dynamic and complex ecosystem that surrounds and interacts with cancer cells within a tumor. It includes a heterogeneous combination of non-malignant cells, extracellular matrix (ECM) components, soluble factors, and physical and chemical conditions that collectively influence tumor behavior, immune responses, and treatment outcomes.1,2 Its complexity, heterogeneity, and suppressive character make it both a challenge and an opportunity in onco-immunology. Understanding TME, especially the critical interactions between cellular and non-cellular components of the TME, is essential for advancing next-generation anti-cancer therapies and achieving durable clinical responses.1,2
Unlike other conventional anti-cancer therapies, cancer immunotherapy (CIT) that has revolutionized cancer treatment paradigms, is based on harnessing body’s immune system to fight cancer. 1 Seminal observations by the pioneering scientists sparked the idea that the immune system could be manipulated to attack cancer cells.3-5 Additionally, the discovery of T cells, along with cytokines like interferons and interleukins, was a crucial breakthrough, as it demonstrated that the immune system is responsible for recognizing and eliminating foreign substances, including cancer cells and leading to tumor shrinkage in some patients.3,6,7 Such observations further fueled research in this area of onco-immunology. Moreover, research on transplanted tumors in mice showed that the immune system could be stimulated to reject these tumors, further suggesting involvement of the immune system in diminishing the tumor burden. 8 These findings, taken together, provided the groundwork for CIT research and development, leading to onset of several first-generation cancer immunotherapy approaches, such as immune checkpoint inhibitors (ICIs), cytokine therapy, and cancer vaccines, with limited success.9-13 Although ICIs revolutionized cancer therapy by releasing inhibitory brakes (eg, PD1, CTLA4) on T cells, a significant proportion of patients exhibit non-responsiveness, relapse, or immune evasion, especially in “cold” tumors with limited immune infiltration. Such limitations have triggered a wave of next-generation precision immunotherapy strategies that combines patient-specific neoantigen discovery, high-resolution spatial immune profiling, and multimodal integration (genomics + proteomics + imaging).14,15 This shift enables context-aware, spatially-informed, and evolution-adaptive immunotherapy approaches, like CAR-T cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, new checkpoint inhibitors, novel cancer vaccines, and oncolytic viruses7-9,14-17 - thus, paving the way toward durable responses even in resistant or immune-silent tumors. Such key conceptual shift from immune checkpoints to immune cartography (often termed as, next-gen immunotherapy), therefore, builds a GPS map to navigate where, what, and how to strike, using neoantigen landscape (what to attack), spatial transcriptomics (where to attack), and cell-cell interaction profiling (how to attack).
Above discussion signifies that knowingly or unknowingly, not only cancer cells but also other tumor-related factors and their surrounding environment have entered mainstream therapeutic interventions, thereby reinvigorating the critical role of the TME in determining the fate of such immune-boosting therapies, also known as immunotherapies. Later on, scientists gained insight into the TME and its impact on CIT by studying the diverse TME-components, including immune cells, stromal cells, and the ECM, using advanced techniques like 3D culture and bioinformatics.10,12 Such advancements in understanding the TME revealed how these components interact and the way their spatial and temporal organization influence immune responses. However, progress in CIT failed to meet the awe-inspiring success that was expected. To mention a few, the number of agents that had showed efficacy in preclinical testing yet failed in early-phase human studies, remains unacceptably high.9,12 Such inconsistencies in large part highlight the pressing need of more effective biomarkers for predicting therapy-response. 11 Therefore, CIT has currently reached another critical inflection point, creating a mandate to both revisit the original challenges and identify new challenges and opportunities with emerging astounding clinical benefit.
Over the years, cancer stem cells (CSCs) have emerged as critical actors and decision-makers in cancer pathogenesis, capable of initiating, maintaining, and repopulating tumors. CSCs are a tiny sub-population within the tumor mass with the ability to self-renew and differentiate into a variety of cancer cell lineages.18,19 In almost every circumstance, CSCs show greater resistance to radiation and chemotherapy than non-stem cancer cells, and given their capacity to replenish the bulk tumor - they posit significant hindrance to the available therapeutic interventions.20,21 Furthermore, CSCs are instrumental in mounting immune evasion, thereby reducing the efficacy of immune-based cancer therapies. CSCs and TME communicate reciprocally, with the latter providing a supportive niche for CSC-survival and self-renewal. On the other hand, CSCs influence in TME polarization and persistence, thereby fostering an immune-suppressive state. As a result, these interactions reduce the effectiveness of conventional cancer therapies, prompting the development of innovative therapeutic strategies to modify the TME by targeting CSCs.20,21
Mounting evidences indicate that the TME is often considered as a barrier to effective cancer treatment. 2 But it is high time to reframe TME in order to leverage maximum therapeutic benefit from it. For the same, instead of attacking the non-stem cancer cells (NSCCs), strategies should focus on targeting CSCs, a critical component of the TME modulating its immune contexture that elicits a barrier to effective cancer treatments.
Here, we provide an update of recent accomplishments, unifying concepts, and future challenges to explore the TME, CSCs, and immunotherapy.
Tumor-microenvironment: The Root of all Paradoxes
Besides tumor cells, the TME contains various immune cells like tumor-associated macrophages (TAMs), dendritic cells (DCs), and other myeloid progenitors, lymphocytes and lymphoid progenitors, as well as other stromal cells like cancer-associated fibroblasts (CAFs), endothelial cells, pericytes, and non-cellular components called extracellular matrix (ECM). These cells infiltrate into the tumor, and therefore, are expected to destroy neoplastic cells.1,2 Therefore, the presence of immune cells in the TME raises a fundamental question, ie, how do cancer cells evade hostile immune attack during initiation, progression, and even relapse? Cytotoxic innate and adaptive immunity should, in principle, regulate and impede tumor progression. 22 Nonetheless, cancer cells possess several countermeasures to evade tumoricidal immune attacks.23,24 Therefore, formation of a tumor is the consequence of a deregulated TME. Such phenomenon, therefore, highlights the fact that tumor initiation is not a singular event, but rather a dynamic immunological conflict between the altered cells and the immune armory.
The immune cells in the TME fall into two categories: (i) anti-tumor or immune stimulant group comprising type 1 helper T cells (Th1), cytotoxic T lymphocytes (CTLs), natural killer cells (NK cells), and monocytes; and (ii) pro-tumor and immune-suppressive group involving type 2 helper T cells (Th2), TAMs, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs).1,2 The tumor immune microenvironment (TIME) is characterized by an abundance of chemokines and cytokines that sustain a balance between pro-tumor and anti-tumor immune responses.1,2 In fact, during initial tumor formation, anti-tumor immune cells accumulate at the site of tumor onset and cause inflammation. However, as the disease progresses, chronic inflammation leads to a pro-tumor immune environment, thereby altering the TME to favor tumor progression. The intricate interactions between cancer cells and other components of the TME create pro-tumor immunological loops that facilitate a conducive environment for tumor survival, progression, and evasion of treatment.25,26
Other than cancer cells, other infiltrating immune cells of the TME, eg, tumor-associated neutrophils (TANs), TAMs, DCs, and mast cells (MCs) activate stromal cells that in turn impart immune-suppressive properties to the TME, facilitating tumor growth. CAFs constitute a substantial portion of the tumor stroma, are activated by plethora of tumor-derived stimuli. They up-regulate immune-checkpoint (IC) markers such as PD1/PDL1, resulting in T cell malfunction and carcinogenesis and emphasizes that the dynamic interplay between cancer cells and the immune niche that can either inhibit or facilitate disease progression, ultimately influencing the efficacy of immunotherapy and various other anti-cancer treatments1,2 (Figure 1). Components of the Tumor Microenvironment (TME): TME Components can be Categorized Broadly Into Two Types. (i) Anti-tumor Group Consisting of Plasma B Cells, Dendritic Cells (DC), Type 1 Helper T Cells (Th1), Cytotoxic T Lymphocytes (CTLs), M1-Subtype of Tumor-Associated Macrophages (TAMs) and Natural Killer Cells (NK Cells) that can Target the Tumor by Tumor-specific Antibody Production, Tumor-Antigen Presentation, Release of Tumor-Killing Cytokines, M1-Subtype of TAM Etc., Respectively. On the Other Hand, (ii) Pro-tumor Group Consisting of Cancer-Associated Fibroblasts (CAFs), Endothelial Cells (ECs), Type 2 Helper T Cells (Th2), Regulatory T Cells (Tregs), Mast Cells, M2-Subtype of TAMs and Myeloid-Derived Suppressor Cells (MDSCs), which Cause Tumor Progression by Releasing Tumor Growth-Aiding Factors, Immune-Suppressive Cytokines Respectively, Thereby Causing Therapy-Resistance and Promoting Angiogenesis and Metastasis. DCs and B Cells, Under the Influence of Tumor, Also Release Factors that Help Cancer Development
Cancer Immunotherapy: A Ray of Hope
Above discussion signifying the altered role of the TME due to cancer and immune cell interplay challenged the scientists to aim at harnessing body’s own immune system to fight cancer. Employing this principle, development of CIT has revolutionized cancer treatment over the past two decades.10,12,27 Conventional anti-cancer therapies (ie, chemotherapy and radiotherapy) aim to kill rapidly dividing cells, including cancer cells - but they do so non-selectively, affecting many healthy cells, especially the immune cells. Therefore, conventional therapies often suppress the immune system as collateral damage, while immunotherapy seeks to culminate and enhance the body’s natural defense mechanisms against cancer.28,29
CIT regimens can be broadly classified into several distinct strategies (Figure 2), which are discussed below. Types of Cancer Immunotherapy (CIT) approaches: CIT Regimens can be Classified Into Several Distinct Strategies. (i) Immune-Checkpoint Inhibitors (ICIs) that Remove Inhibitory “Brakes” on T Cells, Thereby, Restoring Their Ability to Kill Tumor Cells Such as, anti-PD1/PDL1 and anti-CTLA4. (ii) Adoptive Cell Therapies (ACTs) that Involve Infusion of a Large Number of Tumor-specific T Cells that are Engineered or Expanded to Contain Recombinant T-Cell Receptors (TCRs) or Chimeric-Antigen Receptors (CARs), Into the Cancer Patients. (iii) Bispecific Antibodies (bsAbs) that Bridge T Cells and Tumor Cells to Bring Them Near, Thereby Promoting Cancer Cell Killing. (iv) Cancer Vaccines that Train the immune System Towards Recognizing and Attacking Tumor Antigens. (v) Chemokine, and (Vi) Cytokine Therapy, which Boost Systemic immune Cell Expansion and Activation
Immune checkpoint inhibitor (ICI) therapy
IC molecules are essentially inhibitory receptors present on T cells and sometimes other immune cells like NK cells, DCs, and macrophages that are originally meant to protect normal cells from immune mediated-killing. Some well-known examples of IC molecules expressed by the immune compartment include, Programmed Death 1 (PD1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immunoglobulin and mucin domain 3 (TIM3), and Lymphocyte activation gene 3 protein (LAG3). ICI-based therapy which are often monoclonal antibodies, are designed to block the interaction between checkpoint molecules and their ligands. 30 By blocking the checkpoint signal, they remove inhibitory “brakes” on T cells thereby, restoring their ability to kill tumor cells. Clinical trials demonstrate remarkable efficacy in survival and therapy response for melanoma and renal cell carcinoma patients by combination of anti-PD-1 and anti-CTLA-4 therapies.30,31
Adoptive cell transfer (ACT)
ACT is another immunotherapy approach where immune cells like T cells, NK cells, and macrophages from the patient blood, or tumor infiltrating T cells (TILs) are extracted, genetically modified in a laboratory to express ‘chimeric antigen receptors’ (CAR) for targeting specific cancer cell markers or antigens, expanded in-vitro, and infused back into the patient body to fight against tumor.32,33 Therefore, such therapy can be tailored according to individual patient’s specific tumor characteristics, making them a highly personalized treatment option with better efficiency and minimal damage or off-target activities. For example, in B-cell hematological cancers, phase I/II clinical trials have shown positive results with CAR-T cell therapies. 34
Cancer vaccines
This method educates the immune system for recognizing and attacking cancer cells. Vaccines are made from tumor cells, tumor-specific neoantigens, or even from tumor-associated RNAs/carbohydrates/peptides, which instruct the host body to produce specific chemical mediators like antibodies and chemokines that trigger immune response.35,36 These vaccines are targeted to specific proteins or components of cancer cells, enabling the immune system to mount an anti-cancer attack. Several cancer vaccines have shown promising results in clinical trials, with some even gaining FDA approval. Human Papillomavirus (HPV) vaccine prevents against cervical cancer, as well as other cancers like head and neck, anal, and penile cancers. 35 Hepatitis B vaccine protects against the Hepatitis B virus (HBV) that leads to liver cancer. 35 A phase I/II clinical trial demonstrated long lasting immunization effect by treatment with Sipuleucel-T (Provenge), the only FDA-approved peptide vaccine against prostate cancer. 37 Alongside, mRNA neoantigen vaccines have shown promising results in melanoma and kidney cancer, with some patients remaining cancer-free for extended periods. 38
Cytokine and chemokine therapy
Such therapies exploit specific immune-activating proteins, ie, cytokines like interleukins and interferons, or chemokines to enhance the body’s ability to fight cancer.6,39 These cytokines, which otherwise act as chemical messengers, signal the immune system for recognizing and destroying cancer cells, as well as promoting the growth and activation of immune cells like T cells and NK cells. Such therapies also target the cytokine or chemokine receptors to identify and eliminate cancer cells. Examples include the FDA-approved monoclonal antibody Mogamulizumab, that targets CCR4 receptors for treating adult T-cell leukemia. 40 CCR7 ligand CCL21-loaded hydrogels are designed to attract and activate immune cells, like DCs to the tumor site, to augment immune response against tumor. 41 CXCR2 and CXCR4 receptors are involved in tumor cell migration and metastasis. Inhibiting these receptors with monoclonal antibodies prevent cancer cells from spreading and result in reduced tumor growth. Drugs like SB225002 can be used to block CXCR2, leading to a reduction in several gastrointestinal cancer cell viability, 42 while blocking CXCR4 with drugs like CTCE-9908 and AMD3100, lead to reduction in tumor weight or volume in prostate, breast, or ovarian cancer. 43 In fact, combining Trastuzumab (monoclonal antibody targeting HER2 receptor) and CXCR4-targeting AMD3100 can lead to a significant reduction in breast cancer metastasis. 44 Clinical trial showed better response rates when treated with IFNα along with pembrolizumab (anti-PD1) instead of pembrolizumab alone, in melanoma patients. 39
Bispecific antibodies (bsAbs) or bispecific T cell engagers (BiTEs)
These are engineered molecules designed to bind to two different targets, ie, tumor specific antigens and immune cell-specific antigens, simultaneously.45,46 By binding to both the targets, bsAbs bring immune cells into close proximity of cancer cells, enabling them attacking tumor. They also stimulate immune cells to release cytokines, which further enhance anti-tumor immune response.45,46 Pre-clinical studies show high immunogenic and tumor-killing activities using bsAbs in breast cancer models.47,48
Several immunotherapy modalities like cancer vaccines, cytokine therapies etc., are often combined with other therapies like ICIs, to enhance overall immune response. 49
In gist, these therapies aim at modulating the TME thereby overcoming immune suppression, boosting antigen recognition and presentation, enhancing immune cell infiltration and tumor cell killing, as well as creating immunologic memory to prevent even tumor relapse.
Promises yet to be Fulfilled
Although immunotherapy raised high expectations to culminate and enhance body’s natural defenses against cancer, effectiveness of CITs is found to be inconsistent across different cancer types. Success rates of CIT vary significantly depending on the cancer type, stage, and individual patient characteristics. In this context, the predominantly used therapeutic interventions under the broad category of CIT, especially immune checkpoint inhibitor (ICI)-based therapy, patients are typically classified as responders or non-responders based on their clinical and radiological outcomes after treatment.9,11 Responders furnish a beneficial clinical outcome from immunotherapy, such as tumor shrinkage or disappearance of neoplastic mass upon treatment, disease stabilization with no further progression, improved survival metrics (ie, longer progression-free survival (PFS) and overall survival (OS) of cancer patients), augmented immune-related markers like increased T cell activity or favorable shifts in immune profiles, all of which if last for a significant period (months to years) - CIT response is considered durable.9,50 On the other hand, patients who fail to derive meaningful benefit from immunotherapy, are stratified as non-responders.9,50 Such patients not only fail to show reduction in tumor burden, but also are found to be associated with primary or adaptive or acquired resistance (initial response followed by disease progression). They exhibit downregulation of MHC or neoantigen loss, lack of immune infiltration, and presence of immune-suppressive factors in the TME.9,11 One such example is pancreatic cancer wherein, clinical trials with anti-CTLA4 treatment showed unsuccessful outcomes. 51 On the other hand, CAR-T cell therapy has shown remarkable success in hematologic malignancies like B-cell ALL, DLBCL, and multiple myeloma,52,53 but has consistently underperformed in solid tumors.54,55 For example, ACT therapies using CAR-T cells against mesothelin and EGFR showed limited responses. 56 This discrepancy has been a major obstacle in expanding CAR-T therapy beyond blood cancers. The key reasons for this disparity could be categorized into biological, physical, and immunological barriers, as lack of ideal tumor-specific antigens, antigen heterogeneity and antigen loss, as well as the physical barriers and tumor architecture.54,55 For example, hematologic cancers often express well-defined, lineage-restricted antigens, such as, CD19, CD20, BCMA etc., while solid tumors lack truly tumor-specific antigens.52,54,55 Moreover, several targets are also present on normal tissues, thereby increasing on-target/off-tumor toxicity risk.54,57 For example, HER2 is overexpressed in certain breast cancers but also present in lung/heart tissues. 58 Clinical trial data have reported patient morbidity after HER2-targeted CAR-T therapy due to lung toxicity. 59 Alongside, hematologic malignancies tend to have more uniform antigen expression, for example, most B cells express CD19. 52 Solid tumors, on the other hand, are highly heterogeneous in nature— both spatially and temporally. 55 Not only the subclones of solid tumor may lack the desired target antigen(s), but also, they can down-regulate antigens under selective immune pressure. 55 Such factors contribute substantially in determining the efficacy of CAR-T cell-based therapies in solid tumors. Moreover, solid tumors have dense stroma (made up of fibroblasts, ECM proteins like collagen), abnormal vasculature, and hypoxic zones. These factors foster impaired CAR-T cell infiltration and thereby pro-create “immunological cold zones” which cause severe hindrance to the infiltrating engineered CAR-T cells. 54 Hematologic malignancies, by contrast, are diffuse and accessible via circulation. 52 As a consequence, liquid tumors with augmented CAR-T cell infiltration show enhanced efficacy for CAR-T cell-based therapies.
In similar manner, cancer vaccines that prime or boost tumor-specific T cell responses using tumor-associated antigens (TAAs) or neoantigens, face severe challenges identifying truly tumor-specific antigens, thereby eliciting the risk of targeting self-antigens leading to tolerance or autoimmunity.60,61 Additionally, vaccines often fail to elicit strong CD8+ T cell responses as many TAAs are weakly immunogenic or tolerized, as well as in “cold” tumors with low T cell infiltration and high immune-suppressive microenvironments. 62 Further complicating the situation, clonal evolution and neoantigen loss can lead to immune escape and reduced vaccine-efficacy. 63 Alongside, difficulties in optimizing vaccine platforms, dosing, adjuvants, and route of administration are other potential drawbacks in vaccine-directed cancer immunotherapy interventions.60-62 Cytokine and chemokine-based therapy has its intrinsic limitations as well. High-dose cytokines (eg, IL-2, IFN-α, IL-12) can cause severe immune-related adverse events (IRAEs) like vascular leak syndrome or cytokine release syndrome (CRS). 64 Broad immune stimulation may activate regulatory or suppressive immune cells as well, thereby limiting their efficacy. Nonetheless, systemically administered chemokines often fail to redirect immune cells effectively into tumors.65-67 In addition to that, cytokine signaling pathways are redundant, and blocking or enhancing one pathway often leads to onset of the compensatory mechanisms. 68 Moreover, many cytokines are rapidly cleared, requiring frequent high doses, increasing toxicity risks. 69 Bispecific Antibodies (bsAbs)/BiTEs often face collateral damage as tumor antigens (eg, EpCAM, HER2) may also be expressed on normal tissues, causing on-target, off-tumor toxicity. Excessive T cell activation can trigger life-threatening systemic inflammation while prolonged engagement can lead to T cell dysfunction or anergy, especially in solid tumors.70-72 BiTEs (eg, blinatumomab) have short half-life, thereby necessitating continuous intra-venous infusion.73,74 Additionally, tumors may down-regulate or mutate target antigens to evade bsAb-mediated killing 72 whereas, BiTEs face the challenge of poor penetration in solid tumor masses with immune-excluded phenotypes. 75
Worsening the condition, a rare but serious phenomenon called ‘hyper-progression’ is observed where tumor growth accelerates after ICI treatment. Hyper-progression in lung cancer has been well-reported, depicting an increase in tumor growth rate at least about 2 folds during immunotherapy. 76 Such instances were noted in other cancers as well. To effectively assess immunotherapy response, several TME-related parameters should be checked, including the cellular composition, the presence of specific immune cells, and the expression of inhibitory molecules like PDL1 or IDO. 9 Additionally, stromal components and vascularization of the tumor, and other tumor-intrinsic factors like genetic make-up of the tumors, cytokine profile, etc., which can impact the treatment-response, must be evaluated. 11 Careful follow-up and routine imaging are, therefore, of utmost importance for clinical management of such conditions.
Above instances, therefore, highlight the fact that not only tumor cells but also the surrounding milieu, ie, the TME, is crucial in deciding the ultimate fate of CITs.
TME in Shaping Immunotherapy Response
Growing evidences indicate that the TME varies significantly among different cancer types and between patients with the same cancer, thus influencing how tumors grow, evade the immune system, and respond to therapies like immunotherapy. There are several parameters, depending on which the TME across cancer patients vary significantly. Such variation is shaped by tumor genetics, location, tissue origin, as well as host immune system itself. Some of the crucial determining factors are listed below.
Immune infiltration status of the tumors
Cancers can be categorized into three broad immune phenotypes depending on the immune cell infiltration status, different immune phenotypes affecting therapeutic outcomes differently. (i) Tumors (eg, melanoma, non-small cell lung carcinoma (NSCLC)) with high CD8+ T cell infiltration and interferon gamma (IFNγ) expression are termed as ‘immune-inflamed’ or ‘hot’ tumors, and are usually found to be responders.11,77 (ii) The ‘immune-excluded’ tumors like glioblastoma, immune cells present in the TME get trapped in the tumor stroma, thereby failing to penetrate the tumors, and are mostly unlikely to respond to CITs. 11 (iii) The ‘immune-desert’ or ‘cold’ tumors like pancreatic adenocarcinoma (PDAC), prostate adenocarcinoma (PRAD) etc., anti-tumor immune cells are found to be sparse or mostly absent from the tumor mass. However, they often contain immune-suppressive cells like Tregs, MDSCs, and M2 macrophages. These cells actively suppress immune responses and promote tumor growth. In such cases, cancer patients hardly respond to the given immunotherapies, ie, they become non-responders.11,78
Pro-tumorigenic conditions of tumor
Apart from immune cell infiltration ability, the type of immune cells that reside within the TME are also instrumental in deciding immunotherapy outcome. For example, presence and abundance of immune-suppressive cells such as, Tregs, MDSCs, and M2 macrophages correlate with the worse disease prognosis. They vary across different cancers and even patients with same cancer type. For instance, high infiltration of Tregs is often associated with a poor prognosis in several types of tumors, including breast, lung, ovarian, gastric, and pancreatic cancers.79,80 Elevated Treg levels within these tumors are linked to reduced anti-tumor immune responses and a worse OS. 80 Interestingly, there are exceptions like colorectal and esophageal cancers, where high Treg levels are associated with improved survival. 81 These observations suggest that the effects of Tregs on prognosis are not only context-dependent but also rely on the overall nature of the TME of any particular tumor. Such instances, therefore, highlight the importance of the TME in cancer progression and CIT fate determination. Similarly, elevated MDSC counts in the TME lead to various immunosuppressive effects, including suppression of activities of T cell and NK cell, recruitment and activation of Tregs, promotion of tumor angiogenesis and metastasis 82 - all the events being cardinal pre-requisites of resistance to immunotherapy. 83 Therefore, tumor types with high MDSC infiltration, such as melanoma, hepatocellular carcinoma, NSCLC, and breast cancer are often associated with shortened survival and increased risk of recurrence in these cancers.82,83 Likewise, the TME of colorectal cancer, NSCLC, hepatocellular carcinoma (HCC), bladder cancer, esophageal cancer, PDAC, and breast cancer are abundant in M2 macrophage populations.84,85 Studies show that higher densities of M2 macrophages in stroma of the above-mentioned cancer patients correlate with shorter OS and immunotherapy-resistance.86,87
Stromal composition of tumors
This is another important parameter of the TME impacting CIT outcome. 88 Dense fibrotic stroma (ie, desmoplasia, as observed in PDAC) has been detected to physically block the entry of immune cells. 89 Triple-negative breast cancer varies widely in stromal content and immune cell access, thereby significantly differing in responding to CIT interventions.90,91
Cytokine and chemokine profiles
They also play crucial roles in determining the success of immunotherapy by influencing trafficking, survival, and function of immune cells, particularly T cells, within the TME. 6 These biomodulators can promote or suppress tumor growth, mediate T cell exclusion, and affect the overall immune response to cancer. Inflammatory tumors, ie, tumors with high expression of cytokines like CXCL9/10, IFNγ, attract T cells, 92 whereas immunosuppressive tumors secrete TGFβ, IL10, and VEGF, thereby fostering exclusion or dysfunction of immune cells.6,92
Expression of checkpoint molecules
Another crucial factor of the TME is the group of checkpoint molecules that varies widely even within the same cancer type. The presence and levels of immune-checkpoint molecules on tumor cells have significant implications in disease prognosis and predicting therapy response in cancer patients. Some patients may express PDL1,30,93 while others may rely on alternate immune evasion pathways, eg, IDO, TIM3. 30 Furthermore, expression of IC molecules that are dynamic and inducible in nature, can indeed vary over time. Recent reports indicate that the levels and presence of checkpoint molecules on immune/tumor cells change depending on the specific stage of immune response or even by the presence of certain therapies. 93 Such dynamic spatio-temporal expression might limit interpretability of immunotherapy response.
Neoantigen burden and antigen presentation landscape
Tumors with high tumor mutational burden (TMB) (eg, melanoma, NSCLC) present more neoantigens to T cells, thereby promoting heightened T cell responses.94,95 On the other hand, tumors like breast cancer, prostate cancer, colorectal cancer, head and neck squamous cell carcinoma (HNSC), and HCC may lose MHC class I molecules or have poor antigenicity, making them less identifiable to T cells.94,95 Tumors with low antigenicity (for example, prostate, colon, and small cell lung cancers) are more likely to evade immune surveillance and grow unchecked.11,94,95 This can lead to a more aggressive cancer course and a poorer prognosis. Immunotherapies that target tumor-associated antigens may be less effective in treating tumors with low antigenicity, highlighting the importance of understanding the associated factors that contribute to tumor immunogenicity.
Above discussion signifies that the TME is not uniform - it differs depending on cancer type and subtype, and even in individual patient. These differences explain why some patients respond dramatically to immunotherapy while others failing to do so.
Available Biomarkers are Insufficient in Predicting Immunotherapy Success
Above discussion, therefore, portrays that there are multiple factors - both tumor-intrinsic and tumor-extrinsic - that lead to cancer immunotherapy failure, especially ICIs. Such factors are also considered as the biomarkers for immunotherapy response. Tumor-intrinsic factors96,97 include: (i) low TMB, ie, fewer cancer-borne mutations resulting in fewer neoantigen generation, thus providing the immune system with lesser “flags” to recognize the tumor; (ii) defective antigen presentation, ie, tumor antigens are not effectively presented to CD8+ T cells due to loss or mutation of MHC class I molecules or β2-microglobulin gene; (iii) oncogenic pathways governing immune evasion; (iv) Wnt/β-catenin signaling-mediated exclusion of T cell infiltration into tumors; (v) deregulated PI3K/AKT, MYC, MAPK pathways that promote PDL1 expression and/or inhibit apoptosis; and (vi) PTEN loss, which is associated with immune resistance. Another tumor-intrinsic factor includes immune checkpoint negativity or heterogeneity. Tumors that do not express PDL1 are less likely to respond to PD1/PDL1 blockade, 98 and heterogeneous immune checkpoint expression complicates prediction. 93
Various Cancers Along With Their Objective Response Rates to Immunotherapy and Their Respective Immunotherapy-resistance Mechanisms
Cancer Stem Cells Mount Resistance Against Conventional Anti-Cancer Therapies
Tumor initiating cancer stem cells (CSCs) are widely recognized as key drivers of resistance to both chemotherapy and radiotherapy.20,21 CSCs are considered as the source of tumor relapse and metastasis after initial treatment response. Recent reports indicate that even very low number of CSCs are capable of changing anti-tumor milieu into pro-tumor one 116 and can elicit a suppressive immune microenvironment by favoring generation of Treg and other immune-suppressive cells. 117 Their unique biological properties make them significantly more resilient than the bulk tumor population. CSCs are quiescent or dormant in nature, often exist in a non-dividing or slow-cycling state. Thus, such dormant CSCs escape treatment, survive, and later regenerate the tumor mass as chemotherapy and radiation mainly target rapidly dividing cells. CSCs exhibit robust DNA repair mechanisms, and elevated expression of genes like ATM, ATR, BRCA1/2, RAD51, which are involved in DNA damage repair. 118 After radiation that induces DNA double-strand breaks, CSCs repair the damage more efficiently than NSCCs, thereby contributing to radio-resistance. 119 CSCs highly express ATP-binding cassette (ABC) transporters, such as ABCG2 (BCRP), ABCC1, and ABCB1 (P-glycoprotein). These pumps actively expel chemotherapeutic agents from the cell, reducing intracellular drug concentrations and effectiveness, thus correlating chemo-resistance. 120 Another interesting aspect of CSCs is their redox biology, ie, involvement of reactive oxygen species (ROS) as one of the pivotal regulators of stem cell fate. Such CSC-specific redox biology significantly diverges from that of normal stem cells (NSCs). NSCs maintain low ROS to preserve quiescence and genomic stability but are sensitive to oxidative stress. 121 Whereas, CSCs mimic low ROS states but the ROS levels are enhanced with adaptive antioxidant systems, enabling them to resist oxidative damage, survive therapy, promote tumor progression, and escape immune attack. 122 Tumor-initiating CSCs show enhanced anti-oxidant capacity as they maintain lower ROS levels by up-regulating cell’s anti-oxidant system, eg, glutathione, catalase, superoxide dismutase (SOD) etc. 123 Since radio- and chemotherapy work partly by generating ROS to damage DNA, low ROS levels protect CSCs from radiation or chemo-induced killing. Alongside, up-regulated anti-apoptotic proteins eg, SURVIVIN, BCL2 etc., in CSCs prevent programmed cell death of these cells in response to DNA damage or other cytotoxic stresses.21,124 Moreover, these cells are often associated with epithelial to mesenchymal transition (EMT), which directly promotes invasion/metastasis and therapy resistance while indirectly favoring stemness.125,126 Lastly, as a potential member of the TME, CSCs recruit supportive stromal and immune-suppressive cells like TAMs, CAFs etc., that shield them from therapy and promote survival signals via cytokines like IL6, TGFβ etc. as well as through oncogenic Notch/Wnt/Hedgehog pathways.87,127
Such compelling evidences, therefore, portray the instrumental role of CSCs in eliciting resistance against existing anti-cancer therapies.
Cancer Stem Cells: Important Players in Modulating the TME
Above-mentioned instances highlighting the contribution of CSCs in furnishing resistance towards therapies via different mechanisms, as well as the fact that CSCs possess the property of escaping hostile immune system of the host during tumor initiation,128,129 intrigued us to excavate the contributions of CSCs, if any, in mounting resistance against CITs. Recent report from our laboratory indicated that CSCs are responsible for failure of immune checkpoint blockage therapy in pancreatic cancer. 130 CSC-induced generation of pro-tumor Treg cells from CTLs either by shedding TGFβ 116 or by exosomal transfer of Treg cell transcription factor FOXP3, 117 has also been reported from our laboratory. Another group has depicted that not only CSCs show enhanced resistance to T cell cytotoxicity, but also, activated T cell-derived IFNγ directly converts NSCCs to CSCs in breast cancer. 131 Such findings, therefore, uncover an otherwise unexplored concept of immunotherapy-driven induction of stem-like properties in breast tumors.
It is well acknowledged that among the immune components of the TME, TAMs are one of the major invading immune components that can be divided into two subtypes: (i) anti-tumor M1 and (ii) pro-tumor M2 subtypes. A report from our laboratory has discussed that TAMs promote the emergence of CSCs that in turn manipulate the TME by ensuing M1 to M2 switching, thereby creating an immunosuppressive TME. M2-TAM also helps CSCs escaping immune system, thereby raising the possibility of the contribution of CSCs finally in immunotherapy failure.87,132
Role of Different Tumor-specific CSCs in Modulating the TME and Mounting Resistance Towards Immunotherapy Approaches
Cancer stem cells elicit molecular remodeling of the TME
Recent reports highlight that tumor-initiating CSCs manipulate both innate and adaptive immune components, and secrete a wide range of immunosuppressive factors, thereby creating an environment that (i) prevents immune recognition, (ii) promotes immune tolerance, and (iii) inhibits cytotoxic immune responses. CSCs recruit several mechanisms by which they elicit immune suppression in the diseased individuals. To start with, these cells secrete cytokines that directly suppress immune effector cells and promote regulatory/suppressive cell types. Some of the prominent examples are mentioned below.
Transforming growth factor-beta (TGFβ)
CSCs secrete TGFβ, which plays a significant role in immune evasion by suppressing immune cell function, promoting immunosuppressive cells (that further generate immunosuppressive factors), and down-regulating antigen presentation, thereby finally altering the TME. 154 By interfering with the immune system’s ability to recognize and eliminate cancer cells, CSCs therefore contribute to immune evasion and allow tumor development and spread unabatedly.
Interleukin 10 (IL10)
CSCs also evade immune surveillance by secreting IL10, a potent immunosuppressive cytokine.128,155 CSC-derived IL10 is known to act in two different ways. It can blunt DC responses to antigen stimulation, reducing their ability to trigger adaptive immune responses.156,157 Furthermore, this cytokine can suppress the activation and effector functions of T cells, including CTLs and Th1 cells, as well as reduce NK cells’ ability to kill cancer cells.157,158 Such illustrations portray the inhibitory effect of IL10 on the anti-tumor immune cells. On the other hand, CSC-generated IL10 can enhance the immune-suppressive activity of pro-tumor Tregs, leading to further suppression of the immune response. 156 Therefore, by producing IL10, CSCs can actively suppress the immune system, making it less effective in recognizing and targeting cancer cells. This contributes to tumor immune evasion.
Interleukin 6 (IL6)
Secretion of IL6 by CSCs have also been noted that promotes an immunosuppressive TME. 159 IL6 aids in differentiation of T cells into Th17 cells, which can further suppress tumor immunity. It also inhibits the differentiation of Treg cells, which is crucial for maintaining immune homeostasis.159,160 In addition, IL6 recruits and activates MDSCs and M2 macrophages, which are known to suppress T cell functions and contribute to tumor growth. 161 Further complicating the situation, IL6 can up-regulate PDL1 expression on CSC surface, a protein that binds to PD1 on T cells thereby leading to T cell exhaustion and decreased tumor surveillance. 162 Additionally, CSC-shed IL6 can expand CSC pool by converting non-stem cancer cells to CSCs, 163 thus favoring tumor recurrence and metastasis. 125 Therefore, by promoting the production of immunosuppressive factors and cells, IL6 contributes to procreate a favorable microenvironment for tumor growth and survival.
Vascular endothelial growth factor (VEGF)
VEGF acts as a signaling molecule, initiating intracellular pathways that ultimately lead to the activation or modification of transcription factors, thereby regulating gene expression and contributing to processes like angiogenesis, cell survival, and tumor growth.164-166 Interestingly, VEGF can act as a double-edged sword, as it can promote formation of new blood vessels and foster suppression of the immune system.167,168 VEGF stimulates the growth of new blood vessels, ie, neo-angiogenesis and, in turn, vessel maturity and perfusion status significantly influence how VEGF functions and interacts with the immune system.165,169 In tumors, VEGF is often produced in excess due to hypoxia (low oxygen) and other factors, leading to uncontrolled angiogenesis.170,171 This excessive angiogenesis can result in poorly formed and dysfunctional tumor blood vessels. 166 This hinders the infiltration of immune cells, particularly T cells, into the tumor mass.172,173 In contrast to well-perfused vessels that allow proper blood flow, poorly perfused vessels have limited blood flow and can contribute to further hypoxia and VEGF production.165-167,172,174 CSCs are also known to secrete high amounts of VEGF, thereby, promoting tumor angiogenesis and low immune infiltration. 128
Additionally, VEGF can directly suppress the immune system through various mechanisms. VEGF can inhibit the maturation of DCs, which are crucial for initiating immune responses.168,175 VEGF can induce the expression of PDL1 on DCs, which can inhibit T cell activation through interactions with PD1 on T cells. 176 Angiogenic properties of VEGF often promotes infiltration and migration of anti-inflammatory macrophages 177 and MDSCs. 178 VEGF can also promote the proliferation of regulatory T cells (Tregs), which suppress the activity of other immune cells. 179 Thus, by promoting angiogenesis and influencing the composition of immune landscape, CSC-shed VEGF remodels the TME, which has lesser capability of mounting an effective immune response against cancer.
All these evidences point towards the possibility of contributions of immune-evading CSCs in mounting resistance against immunotherapy.
Cancer stem cells remodel the cellular landscape of the TME
Apart from orchestrating immune-modulation and mounting a pro-tumor milieu via cytokine release, CSCs remodel the TME by recruiting and activating immunosuppressive cells like, Tregs, MDSCs, TAMs, and on the other hand, impair the functions of antigen presenting cells like, DC or reduce enzyme-mediated cell-killing potentials of NK cells. Examples of such interactions are provided below.
T cells
CSCs can directly interact with T cells or create an environment that inhibits T cell activation and proliferation.128,129,155 CSCs often up-regulate expression of inhibitory immune-checkpoint ligands such as PDL1, leading to T cell anergy, exhaustion, or apoptosis, thereby dampening anti-tumor immune responses. 180 CSCs have reduced expression of MHC molecules, making them less visible to CTLs. 181 They also resist processing and presenting tumor antigens effectively. CSCs release a variety of immunosuppressive soluble factors like TGFβ, IL10, IL4, PGE2, and other chemokines that directly suppress T cell functions and skew the immune response towards a Th2 phenotype. 129 Furthermore, CSCs can influence APCs, particularly DCs and macrophages, to create an environment conducive to Th2 generation.155,159 These tumor-initiating cells also secrete factors that inhibit proper maturation of DCs. Immature or tolerogenic DCs are less effective at priming robust Th1 responses and may even promote the differentiation of Tregs or Th2 cells. 182 On the other hand, CSCs secrete cytokines (like monocyte-colony stimulating factors, IL6, IL10, TGFβ) that promote differentiation of TAMs into M2 phenotype. 87 M2 macrophages are highly immune-suppressive and characteristically produce Th2-associated cytokines (like IL10 and Arginase), further reinforcing a Th2 environment and inhibiting Th1 responses. 87 Also, CSCs often create a unique metabolic environment within the tumor that influence T cell differentiation.183,184 For example, CSCs often exhibit a high rate of glycolysis, leading to significant lactate production. The acidic, lactate-rich environment can inhibit the function and proliferation of effector T cells (Th1 and CTLs) and potentially favor Th2 differentiation, which might be less metabolically demanding. Additionally, CSCs or associated myeloid cells might express enzymes like IDO (Indoleamine 2,3-dioxygenase)129,185 which depletes tryptophan from the environment and produces kynurenine metabolites. 186 Tryptophan depletion suppresses T cell proliferation, and kynurenine metabolites can promote Treg generation and thereby, potentially influence Th2 differentiation. 187
Tregs
CSCs attract and interact with immunosuppressive Treg cells in the TIME, where CSCs promote expansion and recruitment of Tregs via CCL2, CCL5, and TGFβ.188,189 Recent report from our lab have shown breast cancer CSCs generate immune-suppressive Tregs at early phase of tumor onset by secreting TGFβ. 116 Our group has also demonstrated that breast CSCs transfer FOXP3 protein, one of the crucial markers of suppressive Tregs, via exosomes to anti-tumor T cells for overcoming the delay caused by FOXP3 transcription in the latter cells. 117 Such phenomenon results in generation of pro-tumor Treg cells that suppress the activities of CD8+ T cells and DCs, to finally create a suppressive TME during tumor initiation.129,155,189
MDSCs
CSCs interact with MDSCs to suppress immune system, promote tumor growth and immune evasion.155,189 MDSCs are immature myeloid cells that are activated under pathological conditions, like cancer, and acquire immunosuppressive qualities. CSCs produce factors like granulocyte-macrophage colony-stimulating factor, IL6, and PGE2, to stimulate the differentiation, expansion, and recruitment of MDSCs82,190 whereas, chemo-attractants secreted by other NSCCs can also help recruiting MDSCs to the TME. 191 MDSCs suppress the function of T cells, NK cells, and other immune cells, hindering their ability to attack the tumor, by releasing inhibitory cytokines and chemokines. MDSCs also directly contact and suppress T cells via apoptosis or cell cycle arrest.82,190 Furthermore, MDSCs mount metabolic changes by generating Arginase 1, which depletes arginine needed by T cells, thus creating a more acidic environment. 82 By suppressing immune system, MDSCs create an environment where cancer cells, including CSCs, can thrive. In addition, MDSCs promote neo-angiogenesis,82,159 which is necessary for tumor growth and metastasis. CSCs on the flip side, with their stem cell characteristics, can further enhance the immunosuppressive effects of MDSCs by secreting factors like macrophage migration inhibitory factor, 82 which drive MDSC-mediated immune suppression. Therefore, it is a two-way interaction between CSCs and MDSCs that fosters generation of a suppressive TME.
TAMs
CSCs are also potential interacting partners of TAMs in the TME. They secrete soluble factors, such as cytokines, chemokines, and cell-derived exosomes that attract macrophages to the tumor site. Once recruited, CSCs influence the polarization of TAMs, directing them towards an M2-like phenotype, possessing immune-suppressive and pro-tumorigenic properties. TAMs, on the other hand, also produce immune-suppressive cytokines such as IL10, which can further dampen the immune response. 87 Besides, TAM-derived TGFβ promotes CSC survival and maintains their stem-like properties, contributing to tumor recurrence and chemoresistance. 87 Interestingly, CSCs express high CD47 or ‘eat me not’ signaling molecule, which can interact with receptors on macrophages and prevent them from phagocytizing CSCs. 192 In essence, the interaction between CSCs and TAMs creates a self-perpetuating loop of immunosuppression, where CSCs manipulate the immune system to their advantage, ensuring their survival and contributing to tumor progression.
DCs
Another crucial aspect of CSC-mediated immune-suppression is modulation of DC function by direct interaction with DCs or by influencing the TME in ways that affect DC behavior significantly. CSC-shed chemokines attract DCs to the tumor site or affect their maturation and migration.129,155,189,193 CSCs also release exosomes, which directly interact with DCs, suppress their function, thus leading to a tolerogenic state. 194 DCs can also be modulated by CSC-governed indirect interactions via other immune cells of the TME. For example, CSCs induce the differentiation of MDSCs, which in turn suppress DC function.189,193 CSCs also create a suppressive microenvironment that inhibits DC maturation and activation, preventing them from effectively initiating an anti-tumor immune response.129,155,159 Such CSC-DC interactions result in production of IL6 by DCs, 195 which further promotes tumor growth and angiogenesis. 196 CSCs reshape DCs to produce factors that promote the development and function of Tregs, which suppress anti-tumor immunity.129,197 In melanoma, CSCs have been reported to modulate plasmacytoid DCs to promote tolerance, 198 that suppresses anti-tumor immune responses, allowing tumors to evade immune surveillance and grow unchecked, thereby leading to poor prognosis. Along with the altered TME, DCs can promote tumor progression and metastasis by providing a supportive environment for CSC survival and growth.
In essence, CSCs interact with immune cells within the TME, leading to a potent immune-suppressive state that hinders the body’s ability to fight cancer. Involvement of direct interactions between CSCs and immune cells, as well as indirect influences mediated by CSC-secreted factors creates a vicious cycle that supports tumor growth and resistance to immunotherapies. This is a major reason why tumors with a CSC-enriched niche are more likely to be “immune-cold”, therapy-resistant, and prone to relapse. Therefore, understanding these CSC-immune cell interactions within the TME is crucial for developing new and more effective strategies to overcome cancer and restore a robust anti-tumor immune response.
Role of exosomes in CSC-TME interaction
CSC-Derived Exosomal Contents and Their Immune-Suppressive Properties in Various Cancers
Additionally, mounting experimental and pre-clinical evidences regarding TDEs are gaining traction to be used as easy biomarkers in immunotherapy clinical trials.205,206 There are certain factors that make TDEs valuable in immunotherapy trials, as follows.
Biomarkers for patient stratification and monitoring
TDEs are tumor-specific cargo carrying unique tumor antigens and molecular signatures, like PDL1+ exosomes in plasma correlate with ICI resistance.207,208 In fact, clinical trials like NCT04653740, NCT04258735, and NCT04530890 are exploring exosomal levels as predictors of ICI efficacy in breast cancer. 209 Moreover, miRNA cargo in TDEs reflects immune evasion status or CSC-signatures or activity.210,211 For example, research is ongoing to investigate the roles of exosomal PDL1,212,213 miR-21, 214 or mutant KRAS215,216 as liquid biopsy tools for tracking resistance and predicting therapy response. Thus, tumor antigen–positive exosomes can predict therapy response or tumor burden of the patients.
As antigen sources
TDEs carry tumor-associated antigens (TAAs) like EpCAM, MART1, and HER which are crucial for making cancer vaccines.217,218 Additionally, TDEs have potential to stimulate T cells when loaded onto antigen presenting DCs. Such as, clinical trial (NCT01159288) in NSCLC patients, Dexosome vaccine (DCs pulsed with tumor exosomes) showed safety and modest immunogenicity.219,220 Another such vaccine, DC-TEX is in pre-clinical trials.221,222
Natural delivery vehicles
Being natural cargos, they cross biological barriers easily without mounting immunogenic responses and cell toxicity.223,224 For these reasons, TDEs are being engineered to carry immune modulators, like siRNAs against checkpoint molecules,223,225 pro-inflammatory cytokines,226,227 and TLR agonists. 228 For example, ongoing pre-clinical trials are exploring whether engineered exosomes carrying anti-PDL1 siRNA can reduce immune evasion in experimental settings.229,230
While using TDEs as minimally invasive detection technique, cargo vehicle, and for therapy monitoring present as attractive measure, there are certain risks in exploiting them as well. While utilizing them as delivery medium, it is important to note that TDEs carry immune-suppressive and pro-tumor molecules that could result in suppressing T cells while promoting Tregs and metastasis. 117 On the other hand, TDEs are heterogenous in nature as TDEs from tumors, stroma, and immune cells are difficult to be distinguished. Furthermore, there are some technical limitations too, for using TDEs in clinical trials. Although they are easily isolated from clinical samples or patient-derived tissues, their standardized isolation procedure differs across cancers. Not only that, their quantification and cargo analysis is challenging. It is also difficult to distinguish isolated TDEs from tumors, stroma, and immune cells. For such reasons, their clinical-grade production of TDEs for therapeutic use is not yet robust till date.231,232
Such short-comings warrant standardization of exosome isolation and characterization across malignancies, personalized immunotherapy using patient-specific TDE profiles, synthetic exosome mimetics to avoid immune-suppressive native cargos, and combination strategies, ie, exosome vaccines + checkpoint inhibitors as alternative and effective treatment approach.
Cancer stem cells remodel the epigenetic landscape of the TME
Epigenetic modifications233,234 by CSCs are also crucial for mounting immune evasion. 235 Epigenetic plasticity, by which cells dynamically remodel their epigenome (such as, DNA methylation, histone modifications, and chromatin accessibility) without changing their DNA sequence,236,237 enables CSCs to switch between different cellular states, ie, stem-like and differentiated forms, for adapting to environmental cues and surviving therapeutic pressures.233,234 Such epigenetic changes in CSCs significantly contribute to immune evasion and immunotherapy failure.234,238,239 In fact, stemness-associated TFs are crucial in adopting such specific modes of epigenetic modifications by CSCs to facilitate immune evasion. TFs such as OCT4, SOX2, NANOG, KLF4, ZEB1, and MYC, not only regulate gene expression but also actively reshape chromatin landscape, giving CSCs their hallmark plasticity, immune evasiveness, and therapy resistance.233,238,240 Interestingly, CSC-related transcription factors also drive epigenetic remodeling by recruiting chromatin modifiers to modulate immune inhibitory ligands. CSCs also exhibit enhanced chromatin accessibility at stemness and immune checkpoint loci. Such open regions are TF-rich and TF-dependent.233,241 For instance, stemness TF OCT4 242 and SOX2 243 promote chromatin opening and expression of PDL1. On the other hand, tumor cell-intrinsic PDL1 promotes generation of melanoma and ovarian cancer-initiating cells. 244 Contextually, PDL1 promotes OCT4 and NANOG expression in breast CSCs via activation of PI3K/AKT pathway. 245 MYC activates transcriptional enhancers of CD47 and PDL1 genes. 246 Stemness TFs also form feed-forward loops that amplify self-expression and that of other stemness genes through super-enhancers (SEs). SEs are large clusters of enhancers densely occupied by stemness TFs OCT4/SOX2/NANOG, mediator complex BRD4, and high levels of active epigenetic marks.247-249 Thus, stemness TFs reprogram epigenome to maintain a “cold” immune phenotype by favoring low chemokine secretion, lack of inflammatory gene expression, and suppression of innate immune activation.
Another way of executing immune evasion by CSCs is repressing pro-inflammatory signaling pathways by epigenetic modifications, especially those involving Type I interferons (IFNs), NF-κB, and chemokines, that are otherwise essential for activating DCs, recruiting effector T cells, and sustaining anti-tumor immunity.250-256 In summary, epigenetic modifications in CSCs play a critical role in immune evasion and immunotherapy failure understanding which is, therefore, crucial for developing more effective cancer therapies that can overcome immune evasion and achieve durable responses.
Cancer Stem Cells are Instrumental in Deciding the Fate of Cancer Immunotherapy
Our understanding so far, clearly highlights that CSCs persistently interact with the TME in such a manner that induces immune-suppression as well as immune evasion. The several strategies adopted by CSCs to escape available CIT approaches are discussed below, which can make tumors unresponsive or resistant towards CITs (Figure 3). Mechanisms of Immunotherapy Evasion by Cancer Stem Cells (CSCs): (i) CSCs Escape Therapy-Mediated Killing by Expressing High Anti-apoptotic Proteins (BCL2, SURVIVIN) and Low Death Receptors (Fas, TRAIL). CSCs Evade Macrophage-Mediated Killing by Expressing High CD47. Therapy-Spared CSCs Further Augment Their Pool, Creating an Impregnable Niche. (ii) CSCs Circumvent Immune-Recognition and Priming by Low MHC-I, CD80, CD86 Expression as Well as, Impede T-Cells by Demonstrating High Immune-Checkpoint (IC) Molecules (PDL1, PDL2, Gal9). (iii) CSCs Skew the Tumor Microenvironment (TME) From Anti-Tumor to Immune-Suppressive and Pro-tumor One by Shedding Chemokines and Cytokines (CCL2, CCL5, TGFβ, IL10, IL6) that Cause Anergy in Anti-Tumor immune Cells and Convert Them to Pro-tumor Subtypes
Alteration in antigen-presentation and IC expression
While ICIs aim to reinvigorate T cells and restore immune surveillance, CSCs down-regulate MHC class I molecules, which are essential for presenting tumor antigens to cytotoxic CD8+ T cells. 181 Furthermore, CSCs often lack co-stimulatory molecules, eg, CD80, CD86, which are necessary for proper T cell priming. 257 Mutations or repression of antigen processing machinery (APM) components (eg, TAP, TAPASIN) in CSCs further suppress antigen visibility. 258 As a result, T cells fail to recognize CSCs thus making ICI therapy ineffective.
On the other hand, CSCs overexpress PDL1, the ligand for PD1, even in early tumor stages. Some CSCs are also found to up-regulate TIM3, LAG3, and Galectin-9, which are involved with T cell inhibitory receptors. These ligands persist even when anti-PD1 or anti-CTLA4 therapy is applied, engaging alternative immune escape routes. 93 As a result, even if PD1/PDL1 interaction is blocked, other suppressive pathways remain active, allowing CSC survival and immune evasion. Besides, CSCs secrete a plethora of cytokines such as, TGFβ (promotes Tregs and suppresses cytotoxic T cells), IL10, and VEGF (impair DC function and promote T cell exhaustion), as well as CCL2/CCL5 (recruit MDSCs and M2 macrophages) to employ immune-suppressive cells at the tumor site.129,155,189 Resultantly, TIME becomes hostile to effector T cells, blunting ICI-mediated immune reactivation.
Modulation of the TME
Another interesting aspect of the CSCs is that they are often found in ‘immune-excluded’ or ‘immune-desert’ regions of the TME. Wnt/β-catenin or TGFβ signaling within the CSCs maintain a non-inflammatory TME by (i) suppressing chemokines that recruit T cells 259 or (ii) inhibiting DC infiltration and maturation. 260 Therefore, there hardly remains sufficient T cells to reinvigorate even after ICI-based treatments.7,260 On the other hand, anti-tumor components of the TME, such as effector T cells, NK cells, DCs, and M1 macrophages, which are potential cancer immunotherapy weapons and thus critical for successful immunotherapy interventions by boosting the host immune system, are converted into suppressive, pro-tumor components as a result of CSC activities.129,155,189 Therapeutic efficacy of CITs is heavily dependent on such critical immunological players; hence, inducing their suppressive character defies the ultimate purpose of such therapies. Therefore, immune-activating treatments might eventually boost-up such immune-suppressive TME, preventing them from performing their intended activities.
Escape and enrichment of CSCs after therapy
Furthermore, phenotypic plasticity of CSCs and therapy-induced CSC enrichment make the situation worse. Vulnerable NSCCs may dedifferentiate into CSCs post-treatment, contributing to relapse163,261 or immune escape. 262 Such phenomenon leads to tumor evolution and recurrence even after initial response to ICIs. 262 CSCs also overexpress anti-apoptotic proteins (eg, BCL-2, BCL-XL, SURVIVIN), by means of which they escape T cell-dependent onset of apoptotic events. 21 Besides, they have defective death receptor signaling (eg, Fas, 263 TRAIL-R 264 ) and enhanced DNA repair, 265 making them apoptosis-resistant. Even, CSCs can express CD47 (“don’t eat me” signal), thereby avoid phagocytosis by antigen-presenting cells. 192 Consequently, even if immune cells recognize CSCs, they fail to induce cell death, undermining the therapeutic effect of ICIs.20,21,189,262 Thereby, CSCs employ the above-mentioned mechanisms to act as the key contributors to resistance against ACT, including CAR-T cells, TCR-engineered T cells, and TIL-based therapies. These approaches rely on tumor antigen recognition, T cell infiltration, and cytotoxic killing, all of which CSCs actively evade or suppress.
CSCs lack or down-regulate classical tumor antigens targeted by ACT (eg, HER2, EGFR, GD2). CSCs also exhibit antigen heterogeneity and can rapidly mutate or switch antigens (like CD133 to CD44). Many CSCs show low MHC class I expression, impairing recognition by TCR-engineered T cells. As a result, CAR-T and TCR-T cells fail to recognize CSCs, allowing these cells to persist and drive relapse. 266 CSCs often reside in fibrotic, hypoxic niches that are physically shielded from immune cells. They promote ECM remodeling via secretion of factors like TGF-β, LOX, and VEGF. 267 These niches are often immune-excluded zones with poor T cell infiltration. 268 Therefore, adoptively transferred T cells cannot reach or penetrate CSC niches, leading to incomplete tumor clearance.20,262 As CSCs thrive in hypoxic environments, they display high levels of ROS scavengers (eg, SOD2, CATALASE).123,269 CSCs often rely on oxidative phosphorylation (OXPHOS) or glycolysis, creating a nutrient-depleted and acidic TME.183,184 As a result, ACT T cells become metabolically exhausted, reducing their proliferation, persistence, and killing capacity.
Another cell-oriented CIT intervention is cancer vaccine, which aims at activating the immune system against tumor-specific or tumor-associated antigens. CSCs often remain unaffected towards this therapy, contributing to tumor persistence and relapse. Most cancer vaccines are designed to target differentiation antigens (eg, HER2, 270 MART1, 271 PSA 272 ), which are poorly expressed or absent in CSCs. 273 , thereby limiting immune recognition. 258 Furthermore, antigen heterogeneity enables CSCs to escape immune surveillance after vaccination. Therefore, the immune system fails to “see” CSCs, allowing them to survive immune attack and repopulate the tumor.258,262,274 Additionally, effective vaccination requires antigen presentation via MHC class I/II molecules. CSCs frequently CSCs often lack neoantigens or harbor low mutational burden exhibit MHC class I downregulation and defective APM (eg, TAP, LMP).181,275 Such phenomenon impairs cross-presentation by DCs and T cell priming. As a result, even if a cancer vaccine successfully activates T cells, CSCs remain invisible to them due to poor antigen presentation. Moreover, CSCs reside in protected niches (eg, hypoxic, fibrotic zones, near vasculature) within the TME. They modulate chemokines and ECM components to exclude immune cells, even post-vaccination.262,267 Immune effector cells activated by the vaccine cannot reach CSCs, limiting therapeutic efficacy of cancer vaccines. Cancer vaccines are often limited by their inability to effectively target CSCs. 276 Unless CSC-specific antigens (eg, CD133, ALDH1, EpCAM) are included and the immunosuppressive niche is neutralized, vaccines may only eliminate bulk tumor cells - leaving behind the seeds of relapse.
CSCs are also significant contributors to the failure of cytokine therapies, which are designed to enhance immune system activity through the administration of immune-stimulatory cytokines (eg, IL2, IFNα, IL15, IL12). While these therapies can activate immune cells systemically or within the TME, CSCs resist or subvert these effects through several distinct mechanisms. Even when cytokines stimulate T cell or NK cell activation, CSCs often escape detection due to their immune-privileged phenotype.128,129,155,189 Therefore, cytokine-induced immune responses are redirected against differentiated tumor cells, sparing CSCs. In contrast, CSCs secrete cytokines and chemokines that counteract the pro-inflammatory effects of cytokine therapy, including TGFβ (suppresses CD8+ T cells and NK cells), IL10 (down-regulates antigen presentation and inhibits Th1 responses), VEGF, and PGE2 (suppress DC function).78,191 These anti-inflammatory mediators blunt the effects of therapeutic cytokines, undermining immune activation. Cytokine therapy often relies on enhanced cytotoxic activity of CD8+ T cells and NK cells.6,39 CSCs evade this due to their high expression of anti-apoptotic proteins. Downregulation of death receptors, and up-regulation of CD47, inhibits phagocytosis. CSCs therefore, become intrinsically resistant to immune cell killing - even when those immune cells are hyper-activated by cytokines.128,129,189 While some cytokines (like IFNα or IL12) can promote differentiation, reduce stemness and increase susceptibility to killing, CSCs activate Wnt, Notch, Hedgehog pathways and silence differentiation-associated genes epigenetically, thereby resisting differentiation and preserving their capacity for tumor regeneration and therapy resistance.20,21,260,277 Certain cytokines also support CSC survival, eg, IL6 activates STAT3, promoting CSC self-renewal and survival 278 ; TNFα induces EMT and increases CSC traits. 279 Thus, tumor gains more stem-like properties including resistance following treatment.
The above discussion, therefore, portrays CSCs as a resilient, immune-privileged reservoirs that withstand even aggressive CIT interventions. Unless specifically targeted, CSCs will evade immune recognition, suppress transferred immune cells, and seed tumor recurrence. Hence, targeting CSCs directly or altering the CSC niche is critical to overcoming ICI resistance and achieving durable responses.
Successful Targeting of Cancer Stem Cells is Required to Mount a Permissible Tumor Microenvironment
The preceding section illustrates the fundamental roles of CSCs in facilitating immune evasion, mounting resistance against conventional therapies, remodeling the TME, and contributing to ICI-resistance. Previous reports have demonstrated that targeting CSCs by using small molecule inhibitors like, generic and pharmacologic inhibitors aspirin, mithramycin A, vitamin B6, or miRNAs, results in sensitizing the otherwise impregnable CSCs.163,261,280-282 Such sensitization augments the activities of conventional chemotherapeutics like doxorubicin and facilitates tumor regression. Therefore, targeting or sensitization of CSCs is critically important for achieving long-term success in anti-cancer therapies, particularly when it comes to immunotherapy (Figure 4). While many current immunotherapies (like checkpoint inhibitors) show great initial responses; tumor relapse, metastasis, and resistance are common, and much of these are driven by the survival and immune evasion of CSCs. To device successful and effective therapeutic strategies, we need a multifaceted approach that targets both the intrinsic properties of CSCs and their immune-suppressive interactions with the TME. Targeting CSCs for Effective Therapy Outcome: CSCs can be Sensitized by Multiple Agents (Aspirin, Mithramycin a, Vitamin B6, miRNAs, Etc.) that can Make Them Susceptible to Conventional as Well as Immunotherapy Strategies. CSC-Sensitization Reshapes the TME From Pro-tumor to Anti-Tumor One, Thereby Resulting in High Therapeutic Success
To that end, key methodologies include targeting CSC-surface markers with immune engagement. In this regard, CSC-specific antigens (like CD133, EpCAM, CD44) are exploited to engage immune cells directly.20,21 CAR-T cells engineered to recognize CSC-specific markers, BsAbs or BiTEs to bring T cells to CSCs, and vaccines against CSC-associated antigens to prime T cell responses are used for this purpose.35,36,38 Such methods have the benefit of directing the immune system to identify and eliminate CSCs, thereby overcoming immune evasion. Inhibiting CSC-mediated signaling pathways to remodel TIME, is another such approach of targeting CSCs in order to improve immunotherapy response.20,21,155,159 Checkpoint inhibitors are ineffective in Wnt-driven tumors due to CSC-governed T cell exclusion.259,260 Adding a Wnt inhibitor can create a ‘permissive’ TME, enabling checkpoint therapy to work. On the other hand, blocking TGFβ which promotes CSC niche and immune-suppression, can revert the TIME from suppressive to immune-active form, thus restoring anti-tumor immunity within the cancer patients. As we have already discussed, Notch signaling supports CSC growth and survival, thereby promoting immune evasion involving suppressive immune cells. Therefore, inhibition of Notch might reduce Treg recruitment and enhance function of antigen-presenting cells. Such efforts make the TIME more “immune-permissive” so T cells can access and kill tumor cells by sensitizing CSCs and improve CIT response.
Another potential therapeutic intervention called ‘combinatorial therapy’, aims at reversing CSC-induced immune suppression. To that end, inhibitors or antibodies against CSC-shed immune-suppressive markers like TGFβ, IL10, VEGF, etc., neutralize these signals in conjunction with checkpoint inhibitors (eg, anti-PD1). 49 Such procedures, therefore, overcome local immune suppression, restore T cell function, and boost ICI-based therapy efficacy. Vaccines using CSC antigens (eg, ALDH, CD44, CD133) stimulate adaptive immune responses among the cancer patients. Such therapy trains T cells to recognize and eliminate CSCs before they can cause relapse or metastasis, thereby preventing immune escape and tumor recurrence by CSCs and augmenting immunotherapy response.129,155,189 Last but not the least, combination of ICI + CSC-targeting agents might convert immune-cold tumors to immune-hot, thereby changing the TME nature and improving patient responsiveness.11,49,283 Apart from that, metabolic disruption within CSCs might reduce their resistance properties,183,184 and therefore, enhance immunogenicity for better immunotherapy performance.
Hence, such CSC-targeting tools might break immune resistance, eliminate suppressive immune-privileged niches, and make the TME more accessible to cytotoxic immune cells in the context of CIT. These approaches will constitute a powerful combination of CSC-targeting agents and conventional immuno-therapeutics to improve both initial response and long-term durability of immunotherapy in near future.
Lost in translation: Preclinical Promise vs Clinical Reality in CSC-directed Immunotherapy
Above discussion suggests targeting CSCs with the help of combinatorial and immunotherapeutic means to be a promising approach. Innovative strategies like CAR-T cells, cancer vaccines, and monoclonal antibodies have been developed and further modified to specifically eliminate CSCs. However, clinical and preclinical failures of such therapeutic interventions are well documented due to plasticity, heterogeneity, and immune evasive nature of CSCs.274,284
The major reasons for the failure of CSC-targeting immunotherapies, including concrete examples from CAR-T and vaccine-based strategies are as follows.
The above-mentioned factors indicate the short-comings of successfully translating immune-based therapeutic approaches in clinical settings. Translation of promising results from preclinical drug studies into effective clinical treatments is often hampered by significant gaps between the two stages.
309
These gaps stem from differences in study design, model limitations, and the inherent complexity of human biology. Addressing these discrepancies through improved preclinical models and translational research strategies is crucial for increasing the success rate of new drug development as well as the cancer immunotherapy approaches. Key gaps and challenges are as follows.
Therefore, such pre-clinical/clinical disconnects are the most critical challenges in not only predicting cancer immunotherapy efficacy but also, in cancer drug development. To overcome the clinical–preclinical disconnects in cancer immunotherapy, particularly in targeting CSCs, it is crucial to develop more physiologically relevant preclinical models that accurately mimic the human TME, including immune components and CSC heterogeneity. Additionally, to circumvent the failure of CSC-targeting immunotherapies, innovative strategies need to be devised in order to aim at the core issues like antigen heterogeneity, immune evasion, niche protection, and CSC-plasticity.
Possible solutions to challenges encountered by CSC based-immunotherapy
From the above discussion, it is well-understood that the future of immunotherapies targeting CSCs depends on overcoming these limitations, by the following possible solutions.
Non-uniform and heterogenous expression of target antigens that make targeting of CSCs difficult, can be intercepted by using agents that upregulate tumor antigen expression. For example, pre-clinical study in acute myeloid leukemia showed activation of CD38 by retinoic acid effectively enhanced the efficacy of anti-CD38 CAR-T cells. 331 Similarly, for precise CSC targeting and to lower on-target/off-target toxicities, bi-specific CAR-T cells against two CSC-related antigens can be devised. Pre-clinical mice studies showed that while using anti-CD19 or anti-CD20 CAR-T approaches alone could cause tumor relapse, using dual targeting by CD19-CD20 CAR-T cells induced better response in acute lymphocytic leukemia. 332 Combinatorial treatment using CSC targeted-chemo or radiotherapy with immunotherapy yield better prognosis than monotherapy against CSCs. 333 Anti-CD133 CAR-NK92 cells along with cisplatin treatment was demonstrated to be strongly effective against ovarian CSCs. 334 EpCAM targeting CAR-NK cells along with anti-VEGF agents are observed to be effective in colorectal cancer models. 335
CSCs exist in impenetrable and non-permissible environments. CAR-T cells armed with fibroblast activating protein 336 and heparanase 337 degraded the ECM making it susceptible to T cell attack. Intra-tumoral or local injection of CAR-T cells might also prove to be more efficient than systemic administration. On the other hand, overcoming oxygen deficiency in tumor can be counteracted by external supplementation, which not only enhanced infiltration of T cells and pro-inflammatory cytokines, but reduced immunosuppression like, TGFβ and improved survival. 338 Similarly, it is expected that engineering T cells to withstand nutrient deficiency and acidic environment will make them resistant to exhaustion as well. Depletion of immunosuppressive factors like, IDO1, IL10, along with immune cells such as, MDSCs, Tregs, TAMs using therapeutic agents have shown enhanced cytotoxic effects by CAR-T cells even in solid tumors. 333 Using a TGFβ decoy receptor in CAR-T cells made them impervious to exhaustion in otherwise therapy-resistant prostate cancer. 339
After the T cells have been trafficked to difficult CSC-containing niches, their survival and maintenance becomes imperative for viable outcome. Employing co-stimulatory domains, antiapoptotic factors, cytokines and chemokine-releasing activity are few of the multiple avenues that have been developed to ensure CAR-T persistence. 340 Combinatorial inhibition of ICs by using ICI cocktail (anti-PD1/anti-CTLA/anti-TIM3/anti-LAG3/anti-TIGIT) may overcome ‘checkpoint redundancy’ and T cell senescence. Moreover, CAR-NK cells are being studied to be possibly a better alternative than CAR-T cells owing to being a part of innate immune response and faster action, recognition of multiple markers, ability to recognize cancer populations even with low HLA (such as CSCs), does not require activation by antigen-exposure, can be easily sourced from donors and face lower graft-rejection. 341 Exosomes derived from NK cells have also shown merit due to their smaller structure, thereby improving penetrability and ability to withstand the acidic TME. 300 Similarly, nanoparticles probed with drug conjugates can infiltrate biological barriers in solid tumors, increase delivery efficiency and lower off-target toxicity. A preclinical study furnished nanoparticles with salinomycin and hyaluronic acid could effectively target gastric CSCs. 342
Altogether, combinatorial therapeutic interventions against CSCs mentioned above can solve the challenges faced during CSC based-immunotherapy. However, such treatment tactics would require clear understanding of the disease, using multiple targets, overcoming defenses laid by the TME and attacking NSCCs population at the same time.
Conclusion: Unlocking Cancer Immunotherapy by Targeting CSC-TME Interaction
CSCs are not isolated entities - they are deeply integrated within and supported by the TME. CSC-TME crosstalk fosters a highly coordinated and dynamic immunosuppressive network that enables tumor progression, immune evasion, and fuels resistance to therapies, particularly immunotherapies such as ICIs, ACTs, cytokine treatments, and cancer vaccines. CSCs remodel the TME to favor immune-suppression by secreting immunosuppressive cytokines (eg, TGFβ, IL10), recruiting regulatory immune cells (Tregs, MDSCs, M2 macrophages) that inhibit cytotoxic T and NK cell functions, and by epigenetic reprogramming that foster an anti-inflammatory, suppressive immune microenvironment. Suppressive TME also shields CSCs from immune recognition and attack. Hypoxia, dense ECM, and stromal support (eg, CAFs) create physical and chemical barriers that exclude effector immune cells from reaching CSC niches. Conversely, CSCs down-regulate APM, up-regulate immune checkpoints (eg, PDL1), and resist apoptosis, making them poor targets for immune cells - even when therapies are otherwise effective on bulk tumor cells. Additionally, signaling pathways like Wnt, Notch, and Hedgehog are co-activated by CSCs and the TME components, reinforcing both stemness and immune resistance. Such bi-directional signaling sustains cancer stemness and immune evasion. To enhance the efficacy of CIT, it is imperative to disrupt the CSC-TME alliance, which can be achieved by directly targeting CSCs with surface marker-specific therapies, pathway blockers, and/or by using inhibitors of epigenetic modifications, via reprogramming the TME to reduce immune-suppression, and allow anti-tumor immune cell infiltration, as well as combining immunotherapy with CSC-directed interventions (eg, dual checkpoint and CSC pathway blockade). Restoring antigen presentation and sensitizing CSCs to immune recognition, also results in improving the therapeutic outcome of CITs.
Therefore, disrupting the immune-suppressive dialog between CSCs and the TME represents a transformative frontier in CIT interventions. Only by eroding this protective niche can we achieve durable tumor regression, prevent relapse, and expand the population of true immunotherapy responders.
Footnotes
Author Contributions
Sourio Chakraborty: Conceptualization, literature review and investigation, and writing original draft; Udit Basak: Investigation and writing; Sumon Mukherjee: Scientific illustrations; Sumoyee Mukherjee: Referencing and citations; Tanya Das: Conceptualization, supervision, validation, and final editing. All authors read and approved the submitted version.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the Indian Council of Medical Research (ICMR) Emeritus Scientist scheme (Prof. Tanya Das: 74/1/2020-Pers).
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.