Targeting Tumor Heterogeneity and Epithelial-Mesenchymal Plasticity (EMP) by Anti-Hallmark Combinations, Timing, and Sequencing (CTS) Strategy: Theoretical Basis, Hypothesis, and Proposed Protocol

1. Cancer Initiation, Progression, Transition, Stemness, and Ecological Evolution

1.1. Primary Drivers of Tumor Heterogeneity and EMP

The cascading molecular cellular stress process is the reason for the initiation and progression of cancer; understanding of which becomes essential in targeting and epigenetic reversion of cancer.

1.1.1. ROS – Hypoxia Stress

Due to cellular oxidative stress, excessive accumulation of Reactive Oxygen Species (ROS) is triggered, leading to stabilization of hypoxia-inducible factor-1 alpha (HIF-1α) (Figure 1). Stabilized HIF-1α in this pseudohypoxic state affects mitochondrial and glucose metabolism [13,14]. Figure 1 maps the subsequent events [15]. The onset of hypoxia, downstream effects of aberrant vasculature (including co-option/vascular mimicry), metabolic reprogramming, the acquisition of cancer stem cell traits, the induction of CAFs, and immune dysregulation, leading to cancer heterogeneity with core-resistant phenotypes. Cancer cells maintain a state of "partial EMT," initially characterized by retention of some epithelial characteristics while acquiring increased migratory capabilities, eventually leading to EMT infiltration [16,17]. Hypoxia affects pericyte coverage, promoting the invasiveness of EMT cancer cells [18,19]. With the onset of EMT/EMP/metastasis, secondary mutational resistance sets in [20,21]. Tumor hypoxia also induces angiogenic dormancy, autophagy, and lymphangiogenesis, with increasing therapy resistance [15].

1.1.2. Metabolic and Mitochondrial Stress

The heterodimer of HIF-1α and HIF-1β (Figure 1) promotes the expression of a series of target genes, including glucose transporters (GLUTs) and the glycolytic enzyme lactate dehydrogenase A (LDHA). LDHA accelerates the conversion of pyruvate to lactate, the Warburg effect, facilitating the proliferation of cancer cells [22]. Lactate, a byproduct of glycolysis, diffuses into the tumor microenvironment (TME) extracellularly, leading to an increasingly acidic pH. This increasing extracellular acidosis promotes immunosuppression, rendering several anticancer drugs ineffective. The acidic pH also fosters genomic mutations and further fuels tumor growth and the development of resistant phenotypic subclones (Figure 1) [23, 24. As the metabolic stress increases, proliferating cancer cells run out of nutrients, prompting them to undergo Transforming Growth Factor-beta (TGF-β) - induced EMT, thereby moving away from nutrient-depleted locations [25].

1.1.3. Immune Editing Stress & Avoiding Immune Destruction (Hallmark of Cancer - Enabling Factor)

Immunoediting theory describes a three-step process of immunosurveillance, equilibrium, and immune escape, involving the avoidance of T cell homing and immune destruction [26,27]. Cytotoxic CD8+ T cells initially effectively eliminate proliferating cancer cells during immunosurveillance and later reach equilibrium if the stress increases. The process provides decipherable cues about the dialectics in cross-talk among malignant cells, the Extracellular Matrix (ECM), soluble cues (cytokines/growth factors), and neighboring cells. If the carcinogenesis process continues, within this immune editing loop, Cytotoxic T cells are reprogrammed into immunosuppressive cells that allow cancer cells to evade immune destruction (immune escape), leading to proliferation and the acquisition of greater stemness and therapy resistance [28,29].

1.1.4. Inflammatory Stress and Role of TGF-β

An inflammatory microenvironment (iTME) stress drives carcinogenesis, immune escape, and progression, exacerbated by oxidative stress (ROS). A variety of transcription factors, kinases, and chemokines are involved in this process. The primary factor identified is multifunctional cytokine TGF-β, which initially plays a role in tumor suppression but later becomes fundamental to tumor progression, EMT, and stemness. The less understood “TGF-β switch” activates the Snail and Smad pathways, leading to EMT (Figure 2). In addition, over time, TGF-β acquires multiple tumor-promoting effects, increasing ECM fibrosis and rigidity and preventing immunological cross-talk. Subsequently, cells like CAFs, TAMs, T helper 17 (Th17), Treg cells, and proinflammatory cytokines such as IL-6 and IL-8 sustain EMT [30]. JAK/STAT and NF-κB pathways have also been implicated in maintaining mesenchymal traits and supporting EMT-driven tumor progression [16,17,31].

1.1.5. Spatial Stress (Hypoxia, Compaction, and Nutrient Deprivation)

Proliferation of cancer cells within the given space leads to compressive pressure on each other. Stagnant blood flow, decreased lymphatic drainage, and CAF proliferation, which increase interstitial pressure, create physical barriers [7]. ECM and the cell-stromal communication system change in response to stiffness and altered shear forces. This is an example of mechanotransduction, in which mechanical cues are sensed and transmitted, and these signals are converted into biochemical impulses that promote tumor invasion and metastasis through EMT [32]. The reduction in spatial stress due to treatment intervention and the clearance of dead cancer cells significantly disrupts mechanical barriers and improves ECM softness [33].

Figure 1 depicts the various stress mechanisms that lead to the initiation and multistage, multipathway progression of cancer, including EMT.

1.1.6. Evolutionary Mutational Stress and Therapeutic “Predator – Prey” Game

Secondary resistance can develop during treatment itself, and the predator-prey dynamic could favor the “prey” (cancer), armed with access to the vast information in the human genome [34]. Within the tumor ecosystem, cancer cells exhibit ecological, spatial, and temporal Darwinian dynamics of natural selection, leading to the emergence of diverse cell subpopulations before and after anticancer therapy. The evolutionary changes involve heritable genomic mutations such as chromosomal rearrangements, gene duplication, and aneuploidy [34].

This “evolutionary rescue” can occur even in a small, rapidly declining population of cancer cells, after the lysis and clearance of the bulk of sensitive cells. Secondly, due to the respite these cells receive to recover (for example, due to inappropriate dosing, timing, or a drug holiday). Thirdly, due to exposure to sublethal drug concentrations that facilitate acclimatization (e.g., inadequate drug delivery to the tumor microenvironment (TME)), or following which adoptive drug eflux by cancer cells. At the molecular level, membrane extrusion pumps may amplify suboptimal drug concentrations, leading to multidrug resistance (MDR). A classic example of success in addressing these issues is the consistent sequencing of multiple drugs in pediatric lymphoma to overcome MDR. However, these eco-evolutionary aspects are less thoroughly explored in the literature [34,35].

1.2. Molecular Drivers of EMT-MET Heterogeneity

1.2.1. Key signaling Pathways

These play a critical role in driving preventable EMT processes. Among the most significant is the Transforming Growth Factor-beta (TGF-β) signaling pathway, a well-established inducer of EMT across various cancer types. TGF-β activates Smad proteins, which enter the nucleus and regulate the transcription of EMT-related genes, thereby leading to cytoskeletal reorganization and increased cellular motility [36]. Other targetable influential pathways are depicted in Figure 2. [37,38].

1.2.2. Transcription Factors

EMT-TFs (transcription factors) are instrumental in orchestrating the EMT process. The Snail family (SNAI1 and SNAI2), ZEB family (ZEB1 and ZEB2), and TWIST1 are prominent transcriptional regulators that inhibit the expression of E-cadherin, upregulate N-cadherin, and promote mesenchymal gene expression [39].

1.2.3. Role of MicroRNA

MicroRNA-200c (miR-200c) exerts context-dependent actions that enhance apoptosis, inhibit tumor growth, reduce cellular inflammation, and suppress pyroptosis. In other situations, it can influence EMT promotion, modulate the TME via M2 phenotypic macrophage polarization, the density of TILs, PDL1 expression, CTL exhaustion, and the cancer cell exosomal load. MiR-200c can thus act as both a biomarker and an EMT reversion factor [9,40,41].

1.3. Mechanisms and Impact of MET

Although the exact molecular mechanism remains to be understood, the EMT-MET transition is accompanied by the reestablishment of cell-cell junctions and the regain of epithelial markers, thereby providing an environment essential for regaining proliferation in the new tissues [42,43]. Agents that induce MET can restore epithelial characteristics in cancer cells at the primary site, potentially reduce metastatic potential, and enhance treatment sensitivity [44]. Repressed E-cadherin expression, a hallmark of EMT, is restored upon re-establishment of cell-cell junctions and chromatin accessibility, thereby impairing the migratory capabilities of cancer cells and promoting sensitivity [44]. Reversible surface E-cadherin and cellular gene expression dynamics are closely related to TGF-β levels during an in vitro study [45].

The dynamic interplay between EMT and MET enables them to adapt to various microenvironments throughout cancer progression. Circulating tumor cells have epithelial and mesenchymal markers, indicating a hybrid population. [46,47]. Thus, EMT, when the milieu is stressful (infiltration and metastasis), and MET, when the milieu is fertile (proliferation), are the foundational survival mechanisms by which cancer cells adapt, progress, evolve, and metastasize. In vitro studies show that the addition of bone morphogenetic protein 7 (BMP7), the reduction of TGF-β, and reduced hypoxia encourage MET [48]. EMT-MET connections are spatially separated during the metastatic process and undergo multiple pathophysiological and epigenetic changes, indicating whether MET is truly a reversal of EMT or a transitional process [49]. Therefore, “Reversion” may be a more appropriate term than “reversal”, since cancer cells may not return to their true original state of all epithelial characteristics via MET [10]. During this interim period, MET reversion at the metastatic site can also exhibit higher stemness, being more resistant than that of primary epithelial cells, yet sharing predominantly similar characteristics [47]. Not only MET epithelial restoration, but also normalizing the tumor microenvironment (TME) is a key strategy for cancer reversion, as it restores the functional relationships with normal cells. As recently observed, senescent cells, considered irreversibly phenotypic, can be reactivated to secrete promigratory cytokines or re-enter the cell cycle under detoriating TME [35].

1.4. EMT-MET Spectrum: Epithelial-Mesenchymal Plasticity (EMP) Characterization

The cancer mass has a spectrum of cells spanning the EMT-MET profile, considered epithelial-mesenchymal plasticity (EMP). EMP hybrid EMT/MET features of cancer make it a diverse, shifting target as part of tumor aggressiveness and therapeutic resistance [50,51]. Although EMP accounts for only a fraction of the total number of cancer cells, its complexity is rife with controversy due to a lack of irrefutable evidence. Additionally, several terms dot the literature analysis, like partial EMP (not committed to, thus exploiting both polar states), incomplete EMP (just short of), both referring to not fully transitioning to either way, resulting in a hybrid state (coexpression of both), Transient de-differentiation (EMT)–re-differentiation (MET) switch states, or metastable (relatively fixed somewhere in the EMP axis spectrum). Characterization of EMT and MET, thus, provides invaluable insights into cancer biology [52,53].

Additionally, the interconnected roles of EMT-related transcription factors and cancer stem cell (CSC) markers (e.g., CD44, ALDH) hint at a complex regulatory network that underscores not only the plasticity but also the adaptability of carcinoma cells [42]. Epithelial markers such as E-cadherin, cytokeratins, and occludins are often diminished in advanced cancer stages. In contrast, mesenchymal markers, including vimentin, N-cadherin, and fibronectin, gain prominence, indicating a different profile for evaluation across cancer stages [41,54]. Immunohistochemical profiling of these markers can provide insights into tumor EMT status, assisting prognostic evaluations and therapeutic decisions [55].

The other features in cancer cell evolution include dedifferentiation and redifferentiation, as well as changes in cancer stem cell resetting. The term transdifferentiation refers to another process of MET, in which cells acquire favorable features of different lineages, such as breast cancer cells transforming into adipocytes upon targeted induction [56].

Figure 3. Mesenchymal Epithelial Transition (MET) cancer cell pathways of initiation, invasion, dissemination (1); and seeding/colonization (2); and development of epithelial-mesenchymal plasticity (EMP) (3); reversion to MET of seeded EMT/EMP cells (4a,b) or at primary site (5). DMC = DNA methylation changes; CSC = Cancer Stem Cell; CC = Cell competition – Spatial stress; MCC = Mutated Colorectal Cancer Oncogene; TGFβ = Transforming Growth Factor; DTPs = Drug-Tolerant Persister cells. Created in BioRender. Swamy, K. (2026) https://BioRender.com/0288ysz .

Figure 3. Mesenchymal Epithelial Transition (MET) cancer cell pathways of initiation, invasion, dissemination (1); and seeding/colonization (2); and development of epithelial-mesenchymal plasticity (EMP) (3); reversion to MET of seeded EMT/EMP cells (4a,b) or at primary site (5). DMC = DNA methylation changes; CSC = Cancer Stem Cell; CC = Cell competition – Spatial stress; MCC = Mutated Colorectal Cancer Oncogene; TGFβ = Transforming Growth Factor; DTPs = Drug-Tolerant Persister cells. Created in BioRender. Swamy, K. (2026) https://BioRender.com/0288ysz .

Figure 4. Morphology of Epithelial-Mesenchymal Plasticity (EMP) and topography of vulnerability for restoration of mesenchymal cells with more therapy-sensitive epithelial characteristics - Identifiable targets for Epigenetic modifiers in restoring cell junctions and signaling pathways. TGFβ = Transforming Growth Factor-Beta; TKR = Tyrosine Kinase Receptors; WNT "Wingless/Integrated" family of genes; SHH = Sonic Hedgehog - PTCH1 = Patched-1 Protein - SMO = Smoothened, frizzled family receptor; IL = Interleukin – GP = Glycoprotein. Created in BioRender. Swamy, K. (2026) https://BioRender.com/u5n2djs.

Figure 4. Morphology of Epithelial-Mesenchymal Plasticity (EMP) and topography of vulnerability for restoration of mesenchymal cells with more therapy-sensitive epithelial characteristics - Identifiable targets for Epigenetic modifiers in restoring cell junctions and signaling pathways. TGFβ = Transforming Growth Factor-Beta; TKR = Tyrosine Kinase Receptors; WNT "Wingless/Integrated" family of genes; SHH = Sonic Hedgehog - PTCH1 = Patched-1 Protein - SMO = Smoothened, frizzled family receptor; IL = Interleukin – GP = Glycoprotein. Created in BioRender. Swamy, K. (2026) https://BioRender.com/u5n2djs.

2. Clinical Implications of EMP

1. The presence of EMP post-therapy, compared with pre-therapy, is a poor prognostic factor [52]. Evaluating EMP and stemness before neoadjuvant therapy and in postoperative surgical specimens would indicate an effective/better protocol.

2. Caution needs to be exercised in encouraging EMT to MET transition, as it may inadvertently induce MET proliferation of cancer cells, including the cancer stem cells. Therefore, MET-induction should simultaneously target proliferating MET cells and sensitize cancer EMT/stem cells, underscoring the importance of targeting the epigenetic regulators of interconversion between EMT and MET cellular plasticity in a continuum. The other dimension is to cause extreme EMT reversion to terminal differentiation/apoptosis, which is possible with epigenetic modifiers [6].

3. The advantage of the MET reversion strategy upfront is that it can improve the sensitivity before timed standard therapy CT/RT/Stereotactic Body Radiotherapy (SBRT), and prevent EMT induction by the standard therapies [4,57]. thus targeting the double bind situation of progression by MET versus metastases with EMT.

4. The ECM is as critical as cancer cell features in EMP/stemness and needs to be normalized for better therapy response. The stromal component plays a significant role in MET in addition to intrinsic factors [58].

5. Therefore, all the features of therapy resistance require an appropriate combination, timing, and sequence. Fundamentally, the drug that can sensitize should precede the next one that gets complemented in the scheduled protocol. Additionally, an exploratory analysis is necessary to identify the regimen likely to result in the lowest incidence of partial EMP or DTPs, thereby being most beneficial.

3. Tumor Heterogeneity, CSCs, and EMP – Clonal Cooperation

Cancer cells hide within the compartmentalized TME, with varied types of barriers that provide the ground for the development of multiple layers of resistance to therapeutic interventions. EMP, with its own multifaceted profile, is embedded in this complex, multifactorial network. Additionally, cancer cell-protective dynamic evolutions with unlimited mutational capacity, in which each factor feeds on the others, form a web of therapy-escape-resistant mechanisms (Figure 5 and Figure 6). Additionally, this inhomogeneity can become more complicated with each unsuccessful line of therapy [2,59].

In this highly resilient and dynamic cancer ecosystem, cancer stem cells (CSCs) and EMT are tightly interconnected, with phenotypic switching. EMT can drive a non-stem cell state into a stem cell state, conferring drug resistance [60].

4. Targeting Tumor Heterogeneity and EMP by Combinations, Timing, and Sequencing (CTS) Strategy

The dynamic interplay between EMT, which promotes metastasis, and MET, which drives proliferation, represents a double-bind situation yet a therapeutic opportunity for an advanced, adaptive approach [61]. This biological necessity provides a compelling therapeutic rationale: pharmacologically inducing MET can reprogram aggressive, mesenchymal-like cancer cells to revert to a more differentiated, less motile, and therapeutically susceptible epithelial state primed for timed chemosensitization [62]. The deepest evasive action and the most difficult to eliminate come from DTPs with diverse resistance mechanisms that await the appropriate microenvironmental conditions to re-proliferate [34,63], long after standard therapy ends and presumed cure is achieved. Instead, the pleiotropic, transitioning, and interconversion unstable states should be used to push them toward terminal (trans) differentiation into adipocytes or toward apoptosis, as demonstrated in breast and pancreatic cancers, respectively [6,56,64], using a clear therapeutic window [65] (Figure 7 and Figure 8).

Recent advances in incorporating epigenetic modifiers into treatment protocols could help break this impasse. Appropriate non-cross-resistant combination therapies, in a complementary timing and sequencing (CTS) strategy through personalized monitoring approaches [66], thus exploiting tumor-specific transitional states (EMP) using epigenetic modifiers, state-of-the-art technologies, tumor micro- and macro-environmental immunity-inducing factors, and leveraging advanced artificial intelligence and nanotechnology, form the foundation of this approach. [67,68,69]. Kwon et al. (2021) also detail the “right” timing, combination, sequencing, and delivery of immunotherapy in combination with other treatments. These authors favor using immunotherapy as a priming therapy preceding other anticancer therapies [70].

4.1. Epigenetic Modifiers

4.1.1. Eribulin - A Clinical Proof-of-Concept for Use of Epigenetic Modifiers

The most definitive clinical validation of therapeutic MET induction comes from studies of eribulin mesylate, an FDA-approved microtubule dynamics chromatin remodeling inhibitor used in metastatic breast cancer. Beyond its primary antimitotic function, eribulin possesses a unique, non-mitotic mechanism of action that reverses EMT, normalizes vasculature, reverses tissue hypoxia, and remodels ECM [71,72]. Preclinical work in triple-negative breast cancer (TNBC) models and patient-derived orthotopic xenograft (PDOX) models demonstrated that eribulin causes a profound phenotypic shift from a mesenchymal to an epithelial state. This is characterized by the upregulation of epithelial markers, such as E-cadherin, and the downregulation of mesenchymal markers, including vimentin and the key EMT transcription factor ZEB1. Therefore, to manage this EMT-MET double bind, eribulin can be used as pre-priming therapy before the emergence of chemoresistance and metastatic progression in breast cancer [67,73,74].

4.1.2. Multitargeted-Epigenetic-Therapy (MTET)

The strategy here was to reverse the partial EMT to full MET status by targeting the epigenetic modifiers of the SNAIL, SLUG, and Wnt pathways with a combination of polyphenols to modify epigenomic signatures. The multidrug approach includes GnRH agonists, growth hormone blockers, somatostatin analogs, quinolone-based drugs, the off-label drug Riluzole, several small-molecule TKI inhibitors, C-Met inhibitors to reverse EGFR resistance, and MEK inhibitors targeting various pathways, as depicted in Figure 2 and Figure 4. The authors note that metformin and celecoxib can be context-dependent and should be used cautiously under response monitoring for fear of triggering PI3k, which induces EMT. Plasma VEGF, IGF-1, and TGF-beta 1, imaging, & CTC were used as tumor response markers, with good quality of life in the patients treated [66].

4.1.3. Miscellaneous Epegenetic Modifiers

Histone deacetylase inhibitors (HDACis) can reprogram gene expression to favor an epithelial state [68]. Direct EMT-TF inhibitors restore p53's function in K-Ras-mutated preclinical models [75]. Other compounds, such as Co(III)-Ebox, prevent Snail1 from binding to the E-cadherin promoter, thereby blocking its primary repressive function [76]. In pancreatic adenocarcinoma, suppression of EMT with the nontoxic, low-dose ML210 chemical compound, in combination with gemcitabine, prevented cell migration [77]. In an animal study, systemically administered nanoparticle therapy, which releases siDANCR after endosomal escape at low pH, degrades cellular DANCR through the RNA-Induced Silencing Complex (RISC), thereby preventing EMT and phosphorylation [78]. Repurposed Agents, such as metformin, inhibit EMT via mTORC/TGF-β signaling [79]. Salinomycin, as an AMPK inhibitor, reverses doxorubicin-induced EMT by upregulating E-cadherin and can induce mitochondrial dysfunction and autophagy [80,81]. c-MET inhibitors such as crizotinib resensitize small-cell lung cancer cells to CT by blocking EMT [82]. Restoring miR-200 function using therapeutic mimics can induce MET, upregulate E-cadherin, and reduce motility [83].

4.1.4. General Classification of Epigenetic Modifiers

A growing body of preclinical and clinical evidence has identified several pharmacological agents mentioned above that can be categorized under five categories of epigenetic modifiers: 1. One that acts by preventing EMT and invasive ability [61]. 2. Those that sensitize the cancer cells with MET conversion for subsequent CT/RT/targeted therapy/immunotherapy, requiring proper timing and sequencing in any combination [73,74]. 3. Push the cancer cells to a susceptible EMT/EMP state or lethal ROS when the cancer cells dwell in the DTP, senescence, or dormancy states [84]. 4. Keep the cancer cells frozen “blocked”, in a cell cycle phase away from resistant phenotype evolution [10]. 5. The fifth type is the least explored, reverting the cells to a normalized, evolutionarily suppressed, relatively stable, or benign (trans)differentiation state [10,34]. This emphasizes the mechanism-based classification in the present article that outlines where Epigenetic Modifiers should be integrated into the CTS Strategy.

4.2. Two Concepts for One Strategy

The first conventional concept and foundation of modern anticancer therapy is to eliminate as many cancer cells as possible in the shortest time possible, which is considered to maximize the chance of a cure. Cancer therapies are administered at the maximum tolerated dose (MTD), a foundational concept in phase I clinical trials, and are subsequently translated into other phases of trials, and are a well-established approach. The two premises of MTD approaches are: one, to eliminate cancer cells before resistance develops; and two, that resistant mutations are likely to arise from a large rather than a small population of cancer cells [34,35].

According to the second concept, evolutionary-based therapy (EBT), the conventional MTD approach is the antithesis of Darwinian evolutionary dynamics. According to this proposal, the resistance trait is already encoded as partial resistance at a low frequency in a small subpopulation. When it repopulates after MTD, it progresses into a whole resistance population. In colorectal cancer, a preexisting inherent resistant phenotype has been identified for checkpoint inhibitors, osimertinib, and BET inhibitors. In the second mechanism, cancer cells with phenotypic plasticity, characterized by a spectrum of stem cell, dormant, or poly-aneuploid features, survive the MTD and evolve into a more acquired resistant state. In the evolutionary cascade, gene duplication, loss, and gain-of-function are used to access the entire human genome epigenetically, even in tissues alien to their origin, thereby releasing them from competition with sensitive cancer cells and promoting proliferation. Currently, EBT is in the exploratory phase, focused on monitoring the proliferation of a small resistant population through dose adjustments and epigenetic marker monitoring as part of a dynamic treatment process. In advanced cancers, this approach maintains a good quality of life without toxicity, and no attempt is made to eradicate all cancer cells. The multidisciplinary group implements this approach as an evolutionary-informed therapy option [34,35].

The following perspectives aim to unify these two concepts, based on the understanding that, in principle, they are not mutually exclusive. But they are two dimensions of the complex heterogeneity of cancer.

5.0. HYPOTHESIS – Anti-Hallmark Combinations, Timing, and Sequencing (CTS) Strategy

Hypothesis One: In cancer therapy, normalization of the vasculature and TME is the sine qua non for the predictability of cancer elimination.

Hypothesis Two: Cancer therapy resistance is mediated by tumor heterogeneity, along with epithelial-mesenchymal plasticity, and can be overcome through a well-structured anti-hallmark Combinations, Timing, and Sequencing (CTS) strategy. Additionally, this strategy should deactivate immune-escape signaling in the TME/ECM and restore immune editing by invoking cascading virtuous cycles of antigenicity and adjuvanticity.

It has been more than 26 years since the introduction of hallmarks of cancer by Douglas Hanahan and Robert A. Weinberg in January 2000. They enumerated that tumorigenesis is a multistep process that ultimately culminates in a malignant phenotype, yet it can be simplified to six biological capabilities. Reiterating their concept in 2011, they foresaw “cancer research as an increasingly logical science, in which myriad phenotypic complexities are manifestations of a small set of underlying organizing principles”, despite the charting of clear, detailed intercommunication signaling within tumors in the future [85]. Revisiting the Hanahan et al. principles, we have designed a hypothesis to counter this multistep process using a systematic mult-phase protocol. We want to propose this approach as an anti-hallmark (hallmark-degradation) hypothesis (Figure 7). Table 1 depicts the proposed protocol matching the anti-hallmark concept.

Hypothesis One and related aspects are detailed elsewhere [86]. Hypothesis Two is addressed in the present article, based on the mechanism that counters the specific hallmark of cancer (anti-hallmark mechanisms), thereby helping to place any treatment (present or future) in a particular Phase of the CTS strategy. The heterogeneity across cell populations in various phases of cancer hallmarks necessitates the use of a non-cross-resistant combination at safe, optimal doses [87].

Figure 8 depicts all desirable endpoints in cancer therapy that should serve as the basis for incorporating any drug into the current mechanism-based protocol.

5.1.1. The Prerequisite – Countering Tumor Aberrant Angiogenesis and Hypoxia - Optimum Delivery of the Drug/Activated Immune Cells to the Intended Target Cancer Cell

Due to deficient and aberrant vasculature, increased interstitial pressure, and the mechanotransduction barriers, effective drug delivery into all three compartments of the cancer TME, the cancer cell microenvironment, i.e., intercellular space (CCME), and the intracellular location (ICS) is hindered [7,70]. This patchy distribution of effective drug concentrations results in suboptimal response and invites the evolution of resistant pathways. E.g., Taxanes, which bind to β-tubulin, and anthracyclines, which interact with topo-II, require an effective concentration at target sites (to overcome ABC proteins reducing intracellular drug levels by efflux) [87]. Additionally, loss of stemness by anti-EMT therapy decreases the expression of ATP-binding cassette (ABC) proteins, thereby reducing drug efflux and improving the efficacy of chemotherapy [4].

5.1.2. The Sensitization Flow – Disabling Replicative Immortality and Resisting Cell Death:

In the planned sequencing, the targeted drug and epigenetic modifiers that tend to sensitize subsequent therapy should be placed in the earlier Phase of the protocol. Mesenchymal cells are sensitive to the AXL inhibitor SGI-7079. TGF-β inhibitor SB-431542 or receptor antibodies increase sensitivity to carboplatin. EMT signature showed better control with erlotinib in NSLC but not in other therapies. EMP showed resistance to EGFR and PI3K/AKT inhibitors, irrespective of mutational status. However, when these inhibitors are followed by chemotherapy, they demonstrate greater sensitivity than when either is used alone, in the presence of EMP [88]. Standard anticancer treatments, such as Chemotherapy, targeted therapy, and radiation, can promote oncogenic type III EMT, leading to immune evasion and anoikis resistance via Snail- and Slug-mediated inhibition of p53-induced apoptosis [89,90,91]. After CT/RT, specifically DTPs are believed to constitute a reservoir, eventually leading to irreversible genetic mutations [92]. RT is generally indicated in 50-70% of cancer patients and is known to induce EMT through the TGF-β, Notch, and ERK pathways [93]. RT induced EMT can be overcome with timed epigenetic modifiers. Combining Icotinib with intensity-modulated RT (Trial NCT01534585) in Nasopharyngeal carcinomas inhibits EMT [44] Wang X, Xue. Vactosertib, a TGF-β/ALK5 inhibitor, reduces oxidative stress, fibrosis, and stemness, thereby inhibiting EMT, and also exerts an independent antitumor effect [94]. DNA methyltransferase (DNMT) inhibitor, decitabine, sensitized cancer cells for RT, and resensitized ovarian cancer cells for cisplatin/carboplatin. Bromodomain protein 4 (BRD4) inhibitors enhance sensitivity to chemo-RT and prevent PD-L1 upregulation by RT in NSLC [95]. The synergistic effect of the multimodal approach, combined with advances in nanomaterial-based in situ vaccination technology, awaits proper integration [96].

5.1.3. Managing Immune Destruction Avoidance and Persistent Signaling

Cancer cells employ several mechanisms to evade targeting and immune destruction. One is the transdifferentiation of malignant phenotypes to different embryonic components. In prostate cancer, the development of a hormone-independent neuroendocrine phenotype can be prevented by anti-IL-6 monoclonal antibody siltuximab and JAK kinase inhibitor ruxolitinib, laying the strategic foundation for intercepting EMP at junctional points. Transformed SCLCs in NSCLC respond better to taxanes than de novo ones and are resistant to immunotherapy [97]. DTPs require transitioning from a predominantly MTD schedule to a maintenance schedule complemented with targeted epigenetic reversion therapy. DTPs are vulnerable to bromodomain and extraterminal domain (BET) protein inhibitors, especially when re-entering the cell cycle, which induces lethal levels of ROS-induced apoptosis [84]. The other mechanisms are dedifferentiation/undifferentiation, in which the cells become more aggressive and less responsive to immunotherapy [98]. Conversely, epigenetic forward differentiation of cancer cells terminates malignant proliferation signaling. (Figure 8) [99].

5.1.4. Prevention of Genome Instability and Reduction of Tumor-Promoting Inflammation

It is also essential that any drugs/treatments in the flow “should not leave the epigenetic damage scar,” with altered epigenetic marks influencing inheritable transcriptional effects on surrounding genes. These changes can derail cell identities and could be a reason for late recurrences, making this field a fascinating area for further study. A related phenomenon is therapy-induced stromal senescence, which can induce inflammation in fibroblasts [12,100].

5.1.5. Drugs with Multilevel Antihallmark Action

The critical role of drugs in acting at multiple levels of the pathways, e.g., Eribulin mesylate, a MET inducer, chemosensitizer, and vascular promoter, or TGFβ blockers, with immune-enhancing anti-inflammatory actions (e.g., Vactosertib), has excellent potential in the therapeutic landscape [73,74,94]. Drugs with multispecific and secondary actions that TGFβ blockade would add value to in preventing late recurrences [101]. Valsartan, used routinely as an antihypertensive angiotensin II receptor blocker, has multiple levels of action and blocks TGFβ, ATR1, etc. It acts both as a chemotherapeutic sensitizer and reduces inflammation and ECM deposition, and improves ECM immune cross-talk, thereby preventing fibrosis in animal models [102,103]. In a phase II trial, the other TGFβ blocker, losartan, in combination with FOLFIRINOX, followed by chemoradiotherapy, was clinically beneficial in pancreatic cancer [7]. The other mechanism with potential to impact multiple hallmark levels is restoring “eat me” signals using phagocytosis checkpoint immunotherapy [104].

5.2. Philosophy of the CTS approach

A cure is feasible with the MTD approach in the patient group in which resistant traits have not yet been established. In other cases, phenotypic resistance can evolve during MTD. The current article proposes a CTS strategy that combines both MTD and evolutionary principles to preempt cancer cell resistance, supported by an understanding of the dynamics of cancer cell EMT-MET. The importance of long-term antitumor defense in the simultaneous multi-hit tripartite approach, which reduces tumor load, increases immunogenicity, and reverses immunosuppression, is documented in the literature [105].

This strategy targets the resistant cell population, one after another, in a systematic protocol, with equal focus on epigenetic reversion serving as the backbone of the therapeutic schedule (Figure 7). Simultaneously, downstream oncogenic drivers are eliminated through the logical sequencing of trimodal Chemotherapy, radiotherapy, or immunotherapy along with targeted therapy [97]. The present protocol design simultaneously enhances the immune cycle and the pool of memory cells, restoring/ resetting immune editing. In view of the intensity of combinations, the endpoint should also include minimal or acceptable toxicity. The basis of the present protocol is the mechanism of action of a particular therapeutic modality, and it would be easy to integrate/ substitute any future modality into the protocol based on these principles.

Ros, HIF-1α, and TGFβ are the three primary theoretical targets. However, the limitations lie in the fact that they also play a crucial role in normal body homeostasis, requiring fine targeting. Surgical excision of residual lesions or pulsar/cyclical SBRT boost should be timed to remove/eliminate core DTPs, dormant, and stem cells (Figure 9).

5.3. Supporting Evidence for the Strategy

This hypothesis diverges from conventional approaches by viewing tumor heterogeneity and EMP not as barriers to treatment but as targetable vulnerabilities when approached with appropriate combinations, timing, and sequencing. The Following are some of the existing supporting evidence.

• Combination synergy: TGF-β inhibitors combined with checkpoint inhibitors, which simultaneously block EMT and improve immune cell infiltration [106].
• Importance of timing: Pemetrexed given over a prolonged period before cisplatin prevents EMT-induced resistance, while concurrent administration promotes it [5]. Eribulin before standard chemotherapy sensitizes subsequent chemotherapy [73,74]. Establishing effective multidrug combinations, timing, and sequencing, followed by systematic risk-adapted stratification approaches in clinical trials for pediatric malignancies, is a classic example of a process for controlling cancer and reducing toxicities. [107].
• Clinical evidence: Even with the availability of an extensive list of targeted therapies in lung cancer, the clinical approach is presently veering towards combining with chemotherapy at least to improve the progression-free survival (PFS). The NEJ009 trial showed a higher objective response rate (84%) than that of EGFR-TKI monotherapy (67%), with delayed resistance, manageable toxicity, and improved PFS in advanced (stage III-N2) EGFR-mutated NSCLC.[108]. Improved overall survival was observed with first-line osimertinib-chemotherapy compared with osimertinib alone in the updated phase III FLAURA2 trial, published in January 2026, albeit with an increased risk of reversible adverse events [109].
• Vascular Disruption versus vascular normalization SBRT: A vascular normalization strategy with SBRT is strongly debated against the vascular disruption strategy to this day. The listed criticisms of vascular normalization are: a) The dynamicity of VN and its window period make it challenging in clinical implementation, b) varies with tumor type, c) patient-dependent intratumoral variation, d) dose-dependency (low dose vs moderate dose vs regular dose of AAGs), e) enigma of “normalization window”. However, immunosuppressive signals, including the induction of the TREX1 pathway, HIF-1α, TGF-β, Interleukin-10, and Myeloid Suppressor cells, are acknowledged in the vascular disruption approach [110,111], which may be a critical factor in the present-day immunotherapy-oriented era.

Given the critical importance of reducing hypoxia and improving drug delivery, we propose monitoring serum to detect vascular changes and optimally synchronize AAGs before standard therapies. Also, using this monitoring, we propose, as mapped in Figure 10, the strategy for extending the normalization window program indefinitely. The paradigm-changing concept of vascular normalization was proposed in 2001 by Jain R. K., in the context of the existing concept of vascular disruption [112]. Working on this concept, Goel et al. have provided a reasonable rationale for the normalization window period and its closure, based on patients on AAG drug holidays [113], requiring further refinement. Further, limiting the SBRT dose per fraction to 8 -10 Gy prevents damage to the ECM, preventing the immune suppressive island of epigenetic damage scar recurrence-prone area, whose importance is gaining ground [12]. Further, SBRT doses exceeding 10 Gy per fraction cause endothelial disruption and immune-suppressive ECM changes in a sarcoma model, all of which are unfavorable for long-term control (literally converting SBRT into a palliative type). [114]. With the increasing role of immunotherapy in all cancers, the importance of avoiding epigenetic ECM scars [12] favors a normalization approach that simultaneously retains the immunogenic benefits of SBRT at 7-10 Gy per fraction. Improvement of immune cross-talk not only enhances the efficacy of immunotherapy but also reduces toxicity [115]. Also, cyclical SBRT enhances the long-term “memory cell bank” to prevent recurrence, generating an in situ vaccine effect that can be complemented by in vitro/ex vivo vaccines [Figure 9] [116]. Lesions that no longer respond to immunotherapy combinations may exhibit enhanced antitumor immunity and an in situ vaccine effect, potentially through cancer cell kill, phagocytosis, decreased interstitial pressure, re-oxygenation, and improved drug delivery [7,117]. In the KEYNOTE-01 trial in non-small-cell lung cancer, even at a median of 9.5 months after RT, the immunotherapy group with a previous history of RT had longer progression-free and overall survival [118]. Cyclical SBRT can enhance immunotherapy effects, provided it precedes (rather than follows) each immunotherapy cycle. Clinical benefits of SBRT (3 × 8 Gy) followed by pembrolizumab have been reported in patients with solitary lung cancer metastases [118,119,120]. Induction of immunogenic cell death improves the “hotness” of tumors and immune cell infiltrates [121], Sordo-Bahamonde. This ICD dose range induces mutational neoantigen in the in vivo KP sarcoma model, sensitizing it to immunotherapy when the hot tumor replaces a cold tumor [122].

• It is an opportunity to use a small residual gross tumor as an in-situ neoantigen provider by applying principles of antigenicity and adjuvanticity through local ablative therapies, including SBRT (Figure 8) [123], in select situations.
• Support of advances in monitoring technology: Monitoring therapy with plasma VEGF, IGF-1, and TGF-beta [66]. This is promising for the routine adoption of the proposed protocol. Also, plasma immunopeptidome mining could make it a significant biomarker for follow-up monitoring and ultra-specific therapy targeting method in the coming years [124].

5.4. Methodology of Implementation CTS Strategy & Proposed Protocol

Based on the review above, the proposed strategy comprises FIVE phases that must be implemented systematically. The first step is normalization of the vasculature, which will improve delivery of drugs/cytotoxic cells (to TME, CCME, and ICS) and reduce hypoxia, targeting the primary factor in the genesis of cancer. Other steps immediately follow this. (Table 1).

Several publications highlight the significance of vascular normalization as a precondition for integrating it with other established anticancer therapies, including immunotherapy [131,132,133,134]. Figure 10 illustrates the sum concept of literature to Extended Vascular Normalization across phases I to III [113].

6. Limitations

The perspectives outlined in the CTS protocol presented in this article may fall short of achieving a definitive cure in all patients initiated on this protocol. Essentially, the reversion hypothesis suggests that reversing epigenetic changes and associated pathways in transformed, malignant cells may not restore them to their original normal status. EMP finally leaves residual malignant features in one of the forms, such as DTPs cells, Dormancy, Senescent cells, or “normalized state” (almost normal), which can always have the potential to get reactivated to proliferate and induce recurrence with an evolutionarily more resistant population. Thus, using the CTS strategy to prevent the formation of DTPs, enforcing reversion/differentiation [65], and activating in situ vaccine effects [119] become relevant. A combination-drug intermittent “hit and run” approach strategy is valid in phase V, as senescent cells can take weeks to accumulate again once depleted [149].

Therefore, in addition to the steps outlined in phase V of Table 1, further steps beyond the CTS strategy need to be considered. Finally, when we presume the cancer is cleared and the current routine evaluation indicates no evidence of disease, it is imperative to plan the next level of intelligence against a much more battle-hardened, evolutionarily waiting, hidden tripwire [150]. At this phase, the complexity of managing heterogeneous, profoundly adoptable DTPs, with intricate feed-forward loops, necessitates Systematic Monitoring and Rational Therapeutic targeting (SMART) approaches, which is another subject of in-depth study. The available targeting window is dynamic and should coincide with the detection of entry into or exit from the cancer cell cycle, as detected by yet-to-evolve dependable molecular methods. The evolution of technological breakthroughs in molecular monitoring of the entire spectrum of dormancy (enumerated in the “future directions” sections above) should become a standard [66,144].

6. Future Research Directions to Unravel Complexities and Improve Therapeutic Strategies

Clinical trials are limited in EMP, despite the vast amount of clinical data [142]. Therefore, any effective strategy should include, as enumerated in the present article, the epigenetic modifiers—"currently the most clinically advanced strategy" [61] —as a potential model shift in targeting cancer cell plasticity and tumor heterogeneity. Monitoring plasma VEGF, IGF-1, and TGF-beta levels is promising for adopting Epigenetic modifiers in clinical practice and for evolving innovative approaches that are found effective in the lab [66,101]. Mathematical models are designed given the pivotal role of microRNAs and various signaling pathways in these transitions [143]. The emergence of the ability to evaluate repertoire of antigen peptides in plasma (Immunepeptidomics) offers another explosive frontier for both monitoring therapy and interventional strategies. [144,145]. A comprehensive analysis of these components may reveal potential biomarkers for patient stratification and treatment monitoring proposed in the present protocol. Integrating into the spectrum of these developments, SBRT has the potential to be part of a personalized radiation-in situ-vaccine-immunotherapy accelerated by nanomaterials to overcome the hallmark of genomic instability [146,147].

Single-cell sequencing technologies to map the EMT-MET continuum across different cancer types will provide insights into tumor cell population heterogeneity and the influence of varying microenvironments on these transitional states [37,148].

The lack of routine adoption of EMT pathological characterization, integration into clinical practice, and standardized protocols, despite a vast body of experimental research results and a promising list of drugs, makes its incorporation into the clinical pipeline paramount [[142].

The inexorable advances are in the refinement of trimodal radio-chemo-immunotherapies. The combination therapies are awaiting an aggressive, risk-stratified approach (proven in pediatric malignancies) due to the driving forces of continued technological advances in precision radiation oncology, targeting, and the unraveling of sensitization/resistance molecular mechanisms. [141].

An earlier version of the present article is available as a Preprint.org [151].