Cancer stem cells (CSCs) are a unique population of cells in the tumor that have stemness-like properties, cause drug resistance, potential therapy failure, tumor relapse after treatment, and ultimately decrease the overall survival of the patients [1]. Several theories have attempted to explain the origin of CSC, but none is entirely accepted. One of the factors implicated in the formation of CSCs is chromosomal instability (CIN) [2], which is a defect involving chromosome loss or rearrangement during cell division [3]. CIN may lead to an upregulation of oncogenes or a decrease in the level of tumor suppressor genes [4]. As the tumor grade increases, the rate of occurrence of CIN also increases, and this may lead to the conversion of either normal stem cells (SCs) to CSC or the conversion of non-stem cancer cells (NSCCs) to CSC [5]. Tumors can arise from transformed differentiated cells or transformed SCs [6]. The former can occur during stressful situations like infection, toxins, radiation, metabolic influences, and tissue regeneration, leading to mutagenesis [7]. Typically, complex carcinogenic events are triggered by overexpression of oncogenes, inactivation of tumor suppressors, and cell differentiation by acquiring SC-like characteristics [8].

On the other hand, the transformation of tissue-resident SCs or their progeny requires distinct but few genomic changes for proliferation independent of their niche. Such weak mutational changes have been demonstrated in some cancers, such as gastric cancer, suggesting their resident SC origin [9, 10]. Further, during tumorigenesis, the malignant cells may clonally evolve via the gradual acquisition of genetic changes to enable cancer progression, and some of these may change to CSCs in the process [11, 12]. However, whether the CSCs are present from the initial stages of cancer or formed at the later stages is not well-understood.

CSCs and SC share many common regulatory pathways, including Sonic Hedgehog (Hh), Wnt/β-catenin, and NOTCH, which are crucial for self-renewal [13]. Additional factors like PTEN and polycomb family members influence CSC growth regulation. The CSCs were classically described as a population capable of inducing tumors, especially when transplanted into severe combined immunodeficient mice [14]. Their vital role in tumor initiation and progression makes them ideal candidates for understanding cancer pathogenesis and developing novel therapies [13].

The role of CIN in the modulation of the immune system in tumors remains contradictory. Studies have reported that it can lead to the activation and suppression of the immune system [15]. Recent studies have pointed out how CIN helps CSCs modify themselves and the surrounding environment, thus enabling the malignant cells to evade the immune system [16]. So, in this review article, an attempt has been made to understand the cooperation and interaction between the CIN, CSCs, and cancer immunity (3Cs) in tumors. The review also highlights some of the mechanisms CSCs utilize to evade the immune response with the help of CIN, ultimately leading to the progression of the malignancy. Further, the potential importance of targeting them together in treating advanced-stage cancers is also briefly highlighted.

SCs are undifferentiated cell populations that can self-renew and differentiate into diverse mature progeny [17]. The origin of tumor development from SCs (embryonic rests) was described long back in the nineteenth century [5]. After that, Bonnet and Dick [18] revealed that, like the hematopoietic system, leukemia also has a rare population of cells with stemness-like properties, which he addressed as CSCs. Like leukemia, solid tumors were also observed to contain a similar cellular sub-population capable of self-renewal, differentiation to a variety of cell types, and harboring cancer-initiating properties. Nowadays, CSCs have been reported in many solid tumors, including breast, colon, brain, lung, pancreas, and glioblastoma [19, 20].

CSCs are relatively quiescent cells with the ability to initiate growth and maintain heterogeneity, ultimately promoting metastasis and developing therapeutic resistance in tumors [21]. According to the theory of Paget [22], metastasis involves the detachment of cancer cells from the site of the primary tumor and their spread to distant tissues and organs to establish the secondary tumors. However, this process is imperfect due to the challenging journey the tumor cells must undertake during their mobilization from the familiar microenvironment. CSCs with exceptional survival capabilities, heightened expression of transmembrane transporters, and tumorigenic potential are more capable of enduring stressful conditions in the bloodstream. Thus, they constitute the ideal candidates required for successful distant seeding of the tumor cells [23–25]. CSCs overexpress ATP-binding cassette membrane transporters that lead to high drug efflux, which further contributes to the ineffectiveness of different therapies, including common chemotherapeutic agents such as vinca alkaloids, anthracyclines, and taxanes [13, 26]. Additionally, the CSCs demonstrate an enhanced capability of DNA repair and a high ability to mitigate reactive oxygen species, which may further lead to apoptosis if DNA damage occurs [27]. The above may help them to counter the impact of anti-cancer drugs, which target essential processes in cancer cells like mitosis and DNA repair.

CSCs possess immune evasion capabilities and metabolic adaptations to survive under low nutrient conditions [28]. Further, they demonstrate a high frequency of epithelial-to-mesenchymal transition (EMT). A study on breast cancer highlighted the connection between EMT and SCs [29]. Induction of EMT was observed to elevate the SC gene (CD44^+high/CD24^–/low) profile in the above malignancy [30]. Also, the inhibitors targeting EMT have been noted to suppress SC-like traits. The interaction between EMT and CSCs is believed to play a vital role in relapse, drug resistance, and metastasis [30].

The CSCs express many surface proteins. In past studies, the same have been used as markers to identify and isolate them. The use of these biomarkers in determining the prognosis of tumors has gained broad interest [31]. However, though many of the markers have been listed, a specific marker for each CSC population has not been well-defined. In solid tumors, the CSCs are identified using a panel of cell surface markers like CD44, CD133, epithelial cell adhesion molecule (EpCAM), sex determining region Y-box2 (SOX2), octamer binding transcription factor 3/4 (OCT3/4), NANOG, etc. [28]. Biomarkers for the CSC population found in hematological cancers include CD33, CD44, CD123, C-type lectin-like molecule-1 (CLL1), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), etc. [10].

There have been several fascinating theories regarding the origin of CSCs. Whether they are formed from SCs or cancer cells has been continuously discussed. SCs are pluripotent, so it has been proposed that NSCC gives rise to CSC by upregulating pluripotency-related pathways like Wnt, JAGGED2/NOTCH, and β-catenin [13]. CSCs have been shown to display similar surface and intracellular markers to SCs. However, unlike SCs, the CSCs differentiate into disordered cell types without any role in the functional or structural restoration of the normal tissues. Thus, some authors prefer to label CSCs as SCs with a perturbed differentiation potential [32]. Some studies have suggested that the tumor cells may fuse with hematopoietic SCs, generating multinucleated CSCs with a high migration potential [33]. CSCs also have a high metabolic reprogramming ability, which can switch between glycolysis and oxidative phosphorylation [34].

CIN is the hallmark of most solid tumors and is believed to be an essential factor underlying cancer evolution. It is considered a well-known driver of tumor development and metastasis [62]. It is characterized by the frequent mis-segregation of chromosomes during cell division. The cells with CIN are proposed to have a mis-segregation rate as high as 20% (confer 1% in normal cells) of total cell divisions [63]. The chromosomal rearrangements in tumors may vary from very small to large, ultimately leading to its progression. However, CIN can be predominantly structural or numerical, and distinct types of CINs may exhibit different effects in the case of malignancy. As an example, most aneuploidies are thought to push the cancerous cells towards more aggressive phenotypes. Still, some kinds of CIN may drive the cells towards apoptosis due to extreme imbalance in gene dosage [64]. Malignant cells that have a medium level of CIN demonstrate a poorer prognosis than deficient and high levels [65], indicating that a narrow threshold level of CIN may be essential to facilitate cancer cell survival. High CIN can cause activation of the p53-dependent pathway if the same is active. The latter pushes the cells towards apoptosis and cell cycle arrest [66, 67]. CIN and somatic mutations serve as promoters of genetic heterogeneity and malignant phenotypes in cancer [68]. As malignancy progresses, the heterogeneous malignant cells undergo continual modifications due to CIN, and the novel clones that are fittest to survive are generated. The new karyotypes caused by CIN are responsible for phenotypically and functionally heterogeneous cancer cells co-existing within the same tumor (Figure 2) [15].

Mutations in the cell cycle genes lead to a significant fraction of chromosomes gained or lost during cell division and a change in the structure of the chromosome. Defects in mitotic spindle assembly checkpoint, loss of centriole, error in microtubule kinetochore rearrangement, sister chromatid cohesion defects, defects in DNA repair machinery, supernumerary centrosomes, mitotic plus-end assembly, and cell cycle checkpoints can all lead to CIN [65]. Several qualitative and quantitative approaches have been used for assessing CIN. The visual indicators of CIN include micronuclei, nuclear buds, chromatin bridges, and laggards [69]. These markers of CIN may have different fates, including degradation, chromothripsis, persistence, extrusion, and reincorporation inside the nucleus [70]. It is believed that tumor characteristics and their relationship with the surrounding immune and non-immune microenvironment play a critical role in defining the CIN level conducive to survival and growth.

The TME contains different types of immune cells like natural killer (NK), dendritic cells (DCs), macrophages, and T cells of innate and adaptive immune systems, respectively [79]. The immune system exerts both stimulatory and inhibitory effects on cancers, and the balance of these has profound implications for tumor growth. Cancer immunoediting is defined as the continuous remodeling of the tumor immunogenicity by the immune microenvironment surrounding it [80, 81]. Therefore, the cells involved in cancer immunoediting include the immune cells as well as non-immune cells like the stromal and endothelial cells.

Recent studies have suggested that CSCs may play a significant role in immunoediting [82]. The concept of immunoediting is defined by elimination, equilibrium, and escape phases (3Es) [83]. The first is the elimination phase, defined as the immune system’s ability to recognize and remove cancer cells. CSCs remain relatively secluded in the niches that are the functionally diverse regions within the TME. The niches sustain CSCs, preserve their phenotypic plasticity, help in their protection from the immune system, and contribute to their metastatic potential. Quiescence and low immunogenicity may help CSCs remain protected from a highly functional and active immune system in the elimination phase [36].

In the equilibrium phase, cancerous cells’ growth is limited but not entirely removed. CSCs, due to their higher resistance to immune-mediated killing, may play a crucial part as they can constitute the hidden but continuous source of a few tumor cells that may be present in this phase. In the escape phase, the CSCs may remove all their restrictions and divide asymmetrically. These divisions may give rise to the progenitor cells with a high proliferation rate on one hand and daughter CSCs replenishing their pool on the other hand [84]. By increasing the heterogeneity in the CSC population or the progenitor cells downstream in the hierarchy, CIN may further aid them to escape immune destruction [18, 85, 86]. Also, it has been proposed that CIN may contribute to decreased immunogenicity of CSCs, thus facilitating their escape from the immune system. Overall, a higher CIN will result in more heterogeneity and a lower proportion of the immunogenic antigens of a particular type.

Thus, the threshold required to fire the anti-tumor effector cells may be difficult to reach [87]. CSCs may also modify the microenvironment around themselves, secrete some immune inhibitory factors, and increase the expression of surface inhibitory molecules like PDL-1 [88, 89]. The CSCs can secrete a variety of molecules, such as TGF-β, IL-6, IL-10, IL-4, and prostaglandin E2 [90], and also show improper antigen presentation that helps in their protection from both innate and adaptive immune systems [80, 91]. A lower level of CSC immunogenicity as compared to NSCC has been reported in previous studies [89]. The representatives of CIN, like micronuclei, may rupture to release DNA into the cytoplasm, which is recognized by nuclear DNA sensors like cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) to initiate a stimulator of interferon (IFN) genes (STING)-mediated type-1 IFN response. The same may lead to inflammation [92]. However, prolonged inflammation via this pathway may further enhance CIN and promote tumorigenesis. Also, the cGAS-STING pathway may be downregulated in some viral-induced tumors or by some molecules like excision repair cross complementation group 1 (ERCC1) in tumors [93]. Nevertheless, the cGAS-STING pathway is proposed to play a vital role in cancer immune responses, which may be governed by factors like the type of tumor and strength and duration of its activation [94]. Moreover, the CIN-activated cGAS-STING pathway may impact CSCs. A study by Liu et al. [95] reported the potentiation of cancer stemness by intrinsic activation of cGAS-STING via increased STAT3 (Figure 3).

Modern studies have highlighted the importance of the above 3Cs in carcinogenesis, however, in isolation. The level of CIN is determined by microscopy-based identification of morphological markers like micronuclei or hybridization techniques like fluorescence in situ hybridization or comparative genome hybridization. Similarly, past studies have used designated CD markers to identify stemness or the nature of immune response in cancer [119]. Choosing the most relevant panel of markers and assays for gauzing the 3Cs in the tumor needs to be undertaken in future studies. Further, techniques like flow cytometry or real-time polymerase chain reaction (PCR) can then be explored to assess these markers in the routine clinical setting for diagnosis and to decide upon the precision therapies from the existing or novel drugs. For example, drugs targeting multiple aspects of CSCs, whether they are the surface molecules or the signaling factors related to them, are being extensively investigated.

Similarly, the knowledge that CIN beyond a certain level is lethal to cancer cells is being used to develop novel therapies (Table 1). The immune response dynamics in cancer have also led to a torrent of immune-based therapeutics like antibodies, chimeric antigen receptor (CAR)-T cells, cytokines, immune checkpoint inhibitors, etc. However, despite so much progress, the treatment of advanced cancers remains problematic. At present, different types of approaches, ranging from gene therapy to small interfering RNAs (siRNAs) to novel compounds, are being used with or without conventional chemotherapy to achieve complete elimination of cancer. CAR-T and CAR-NK cells designed against the molecular markers overexpressed in CSC, like EpCAM, CD133, CD44 isoform variant 6, etc., are being investigated to treat certain cancers (phase I clinical trial) [120]. So, searching for a therapeutic strategy addressing the 3Cs together may be the elixir of life for difficult-to-target cancer patients. The above may involve a single compound or combination of drugs simultaneously targeting stemness, CIN, and disadvantageous immune factors in one go.

The ensuing discussion proves beyond doubt that the 3Cs mentioned above constitute the crucial three pillars of carcinogenesis. Although 3Cs have been observed to influence each other in one or multiple ways, the pathways and molecules governing their interactions are complex and incompletely understood. The association between the 3Cs constitutes a vicious cycle that drives the cancerous process forward. Understanding the role of these, especially in the context of each other, may help in identifying a panel of biomarkers for early detection and therapeutic targeting of malignancies. Recent studies have drawn attention to the importance of modulation of CIN-induced cGAS-STING pathway and subsequent immune response in cancer treatment. Similarly, PI3K-AKT inhibitors may be used to target all three events together. CAR-based immunotherapy is being investigated to target surface markers of CSCs to kill them specifically. Thus, the effective targeting of the triad of CIN, CSCs, and associated hostile immune microenvironment together carries plenty of potential for improving the therapeutic outcome of highly aggressive, metastatic, and drug-resistant tumors with poorly defined molecular targets in the future.