Some researchers are adopting a different strategy rather than depending on endogenous signals, which come from the body. They are tricking a tumor into secreting synthetic biomarkers that can be detected in biofluids while the tumor is still undetectable by any existing technique by using cunning engineering technologies and tumor-specific biological knowledge. When ingested, the exogenously supplied bioengineered sensors can send out a signal indicating the presence of cancer cells. These techniques have successfully detected significantly lower tumor sizes in animal models (28). The biological, physiological, and statistical constraints of endogenous biomarkers serve as a justification for the development of synthetic biomarkers. Endogenous biomarkers such as proteins in the pool of blood and having varying secretion rates are difficult to detect due to short periods of retention and frequent clearance from circulation (29). These represent a new class of diagnostics that use bioengineered sensors such as molecular probes or genetically encoded vectors that take the advantage of the potentially dysregulated characteristics of early stage tumors or their precursors which could become lethal, inside the body to scan for early stage tumors and amplify illness signals to levels that may be greater than those of shed biomarkers detectable from body fluids such as blood and urine. Several imaging techniques also employ synthetic biomarker approach including reporter gene imaging, in which an exogenous molecular tracer (such as a positron emitting probe) is systematically infused (29). Synthetic biomarkers on the basis of their activities are called protease-activated synthetic biomarkers that are particularly effective molecular amplifiers. Apart from it vector-based, mammalian cell-based, and bacterial cell-based synthetic biomarkers are also employed on the basis of their advantages (Figure 1). Moreover, some preclinical studies have reported the potential use of activity-based sensor composed IONPs synthetic biomarkers for early detection of LS174T colorectal cancer (30).

Tumor cells in circulation: According to a recent study, a limited fraction of circulating tumor cells (CTCs) with the ability to cause metastasis include those that express the molecules; EpCAM, CD44, CD47, and MET. It has been found that individual CTCs from the same patient have different KRAS, BRAF, and PIK3CA mutations. CTC are detected using flow cytometry and immunocytochemical analysis are also highly sensitive methods to detect biomarkers for CRC (33).

Transcription factors: The caudal type homeobox transcription factor (CDX2) is one of many transcription factors that contribute to the development and differentiation of the intestine (41). It is a widely used immunomarker for CRC as it is a tumor suppressor gene in CRC and its expression is lacking in CRC cases. Another transcription factor special AT-rich sequence-binding protein 2 (SATB2) regulates skeletogenesis and is a CRC biomarker with a positivity rate of 83.7% of stage III/IV colorectal adenocarcinomas, 91.4% of stage II and 92.4% of stage I of this malignancy (42).

As the exfoliating tumor cells first occur in the large intestine or rectal lumen during colorectal carcinogenesis, stool specimens are more suitable for the early identification of CRC than blood samples (48). The presence of stool biomarkers has resulted in the early detection of CRC. The guaiac-based fecal occult blood testing (gFOBT) and fecal immunochemical test are mostly used for the screening of rectal blood loss, which is the biomarkers in the stool. The fecal microRNA-106a test is used to detect mRNAs in stool (37). Tumor suppressor genes are rendered inactive by hypermethylation at every stage of carcinogenesis, from polyps to colorectal adenocarcinomas. Many genes, particularly those in the promoter region, are methylated in CRC, including APC, MLH1, MGMT, SFRP1, SFRP2, CDK2A, TIMP3, VIM, SEPT, CDH1, and HLTF (49). There are numerous methylated DNA stool biomarkers used in CRC like SFRP methylation, CDKN2A, MGMT methylation, Vimentin methylation, NDRG4 methylation, BMP3 methylation, K-ras mutation, hypermethylated SCNA, etc.

Early detection and screening for CRC is necessary and could potentially be lifesaving in many cases, as the symptoms of CRC often tend to develop late in the natural course of the disease (50). Multiple methods can be used to screen and perform early diagnosis of CRC, each with its associated advantages and disadvantages. The most important feature of these tests is the test’s sensitivity, and to a certain degree, its specificity (51).

The need for more specific and sensitive biomarkers to detect CRC arises from the fact that CRC is one of the top four most prevalent cancers worldwide (71) with a high mortality rate. The current non-invasive techniques used for screening, for example, are not very sensitive to the earlier stages of cancer and may miss any pre-cancerous lesions and polyps. According to Imperiale et al. (72) “In asymptomatic persons at average risk for colorectal cancer, multitarget stool DNA testing detected significantly more cancers than did FIT but had more false positive results” (73). The finding further establishes the need for more sensitive biomarkers along with the already used screening techniques. The emergence of gene expression analysis along with transcriptome studies has allowed scientists to categorize CRC into subtypes for developing a better understanding of the disease and for devising better treatment strategies based on the subtype of CRC a patient may have. (74)Maida et al. (Maida et al., 2017) performed Molecular sub-typing of colorectal cancer, dividing it into 4 major subtypes: CMS1, CMS2, CMS3, and CMS4. This analysis has also elucidated new biomarkers. Similarly, Multi-Omics studies analyzing large amounts of data on the structure and function of several biological molecules in their totality have led to a better understanding of multifaceted and complex diseases like cancer. The omics studies including genomics, transcriptomics, proteomics, metabolomics, glycomics, etc. have revealed many new promising biomarkers for CRC. These biomarkers include several different kinds of molecules which may be DNA–RNA-based, protein-based, metabolite-based, or even volatile substances found in a patient’s breath. They can be detected utilizing techniques like genomic analysis, mutation analysis using hybridization arrays, micro-arrays, bioinformatics analysis, mass spectroscopy, Gas- Chromatography MS, Gel electrophoresis, etc. (9).

Acute inflammation, also known as resolved inflammation, is a self-limiting adaptive host defense mechanism that brings the body back to a state of homeostasis. However, persistent, unchecked, or unresolved inflammation can result in a number of diseases, including cancer. Nonsteroidal anti-inflammatory drugs (NSAIDs), like aspirin, lower the risk and death from several malignancies, which is significant evidence that connects inflammation and cancer. Clinical studies using COX-2 inhibitors for cancer prevention or therapy were justified by the overexpression of COX-2 in the colon and many other malignancies. NSAIDs, on the other hand, do not need COX-2 to prevent cancer (79).

Since ancient times, it has been understood that the primary reaction to damage is “Inflammation.” Hippocrates, a Greek physician, may have been the first to view inflammation as the start of a healing process and used terms like erysipelas and edema to characterize its symptoms (80). The body’s reaction to an exposure, such as an infection or an injury, is inflammation. NSAIDs have been identified as the prototype chemopreventive drugs against several types of cancer by more than 30 epidemiological investigations that combined reported findings on more than one million participants. NSAIDs can affect the microenvironment of tumors by slowing cell migration, boosting apoptosis, and decreasing chemosensitivity. Targeting the molecules (COX-2 cyclooxygenase 2, NF-kB, VEGF) involved in the inflammatory process might offer a useful technique for cancer prevention and therapy since they can predispose to tumors (81). Several NSAIDs like aspirin, celecoxib, piroxicam have shown preventive effects on inflammation in colorectal cancer. Colorectal cancer-related prevention by NSAIDs mostly works by acting on the pathway of the eicosanoids (82). NSAIDs have been shown in the past to have anti-tumor effectiveness, less toxicity, and non-specific side effects than those caused by conventional chemotherapy. They were also able to limit tumor growth by causing changes in the inflammatory environment of the tumor (83). NSAIDs have demonstrated chemoprotective and anti-inflammatory effects on inflammations associated with tumors. The fact that COXIB has more notable protective advantages than non-selective NSAIDs against a variety of malignancies is associated with a larger reduction in the risk of cancer (84, 85).

The drugs which lower cholesterol, also known as statins are made of tiny molecules called 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-coA) reductase inhibitors. Since statins show anti-carcinogenic characteristics in several in vitro and in vivo preclinical tests, there is a great interest in finding out how they might be used in cancer chemoprevention. Statin use may offer some preventive benefits against total cancer risk, according to some observational human research, but not others (113).

Statins are routinely used to reduce cholesterol and NSAIDs are mainly used to treat inflammation. Recent studies have focused on their potential function as cancer chemo-preventive drugs. Human studies have not shown solid data on the protective benefits of statins against various malignancies, although NSAIDs have yielded more compelling results for cancer prevention, particularly in CRC. Combining statins with NSAIDs may induce synergy and result in a reduction in the doses needed for each agent, which is a potential technique for improving cancer prevention effectiveness. This method is of particular importance for the prospective long-term utilization of low dosages of NSAIDs and statins for cancer chemoprevention. Significantly, colorectal cancer chemo-preventive studies have shown elevated possibility for gastrointestinal and cardiovascular adverse effects linked to NSAID usage. A growing body of research has conclusively shown that NSAIDs help prevent cancer, particularly colorectal cancer. Because of the potential elevated risk of severe cardiovascular and gastrointestinal side effects, relatively high dose needed to produce the observed chemo-preventive benefit in human studies can dissuade the long-term usage of NSAIDs alone for cancer prevention (114). Emerging research suggests that combining cancer chemo-preventive drugs, NSAIDs with distinct mechanisms of action may result in synergistic interactions, which might result in far higher anti-carcinogenesis benefits than each chemo-preventive agent could independently. NSAIDs have demonstrated synergistic effect in various other in vitro studies when treated with other therapeutic agents for example EGFR family inhibitors, statins, TRAIL receptor ligands, and PPARg ligands (115).

The combined use of statins and NSAIDs is particularly intriguing for cancer prevention. Atorvastatin is an example of the drug that was prescribed most in the year 2006 in US. In a significant experiment, to examine the results in individuals with coronary artery disease, pravastatin usage was found to be linked to a 43% decrease in several newly detected instances of colon cancer. Notably, 83% of individuals in both placebo and pravastatin groups received aspirin every day, implying that interaction between aspirin and pravastatin may have an improved protective impact (116).

The effects of statins and aspirin on risk of CRC were studied in a population-based case control research (117). This study comprised 612 controls and 537 patients with CRC cases that had been histologically proven. Frequent use of aspirin at a low dosage level was linked to a moderate reduced risk for CRC, whereas frequent use of statins, primarily simvastatin and atorvastatin, was linked to a stronger risk reduction. The most intriguing finding was that taking statins and lower dose of aspirin together for 5 years or more was linked to 62% risk of risk in CRC.

Utilizing the AOM rat model, effectiveness of celecoxib, aspirin, and atorvastatin against colon carcinogenesis when given separately on high dosage levels and when combined at low dosage levels (118). In comparison to single high doses of atorvastatin given at 150 ppm or celecoxib given at 600 ppm, the combination of 100 ppm atorvastatin and 300 ppm celecoxib reduced the prevalence and multiplicity of adenocarcinomas. Accordingly, low-dose combination of atorvastatin and aspirin significantly inhibited the prevalance and multiplicity of adenocarcinoma when compared to higher doses of each treatment alone. The effects of celecoxib and atorvastatin was examined on growth of adenomatous polyps in intestines in a different experiment utilizing the ApcMin/+ mouse model. Combining atorvastatin and celecoxib at 100 ppm and 300 ppm, respectively, was found to completely suppress colonic adenomatous polyps and reduce adenomatous polyps in small intestines by 86%. However, these effects were more potent than those brought on by either celecoxib or atorvastatin treatment administered separately (119). Together, these findings certainly showed that statin/NSAID combination regimens significantly increased the effectiveness of either type of agent administered alone in preventing cancer. This strongly supports the use of the statin/NSAID combination as a promising method for cancer chemoprevention.

Studies on the detection of colorectal cancer and its prevention are currently an expanding area of clinical oncology because it is one of the most prevalent tumors in the world. An analysis of randomized controlled, double blinded clinical studies including a few NSAIDs such as aspirin, sulindac, and celecoxib and colorectal cancer chemoprevention was done for this study. People taking NSAIDs had a decreased incidence of CRC, which points to the medications sustained chemo-preventive effects in both per-clinical and clinical studies. This advanced method of treating colorectal cancer could make it less fatal and more manageable. Clinical trials have examined and analyzed different NSAIDs for their proper dosage, duration, and therapeutic effects on CRC chemoprevention (110, 132, 133). Evidence from these clinical trials determined the extent of their chemopreventiveness in CRC. Seven trials on the use of aspirin in monotherapy, polytherapy with folic acid or eicosapentanoic acid for the prevention of CRC has been completed to date. For aspirin one such study involved the people with a history of CRC and not the ones with FAP or HNPCC. Patients had to wait for at least 5 years following tumor removal before experiencing a relapse to be eligible for carrying out colorectal adenoma prevention study (CAPS). It was found that the groups receiving aspirin 325 mg per day for 3 years had reduced average number of adenomas recurrence by 35% (134). Similar encouraging results were reached in the Asian population in the clinical trials with ASA 100 mg/day for 2 years, which involved participants with adenoma and a history of colon cancer (135). The Rothwell team also looked at if there was any weight or height dependence and how aspirin affected the risk of colon cancer over the next 20 years. In people weighing 70 kg or more, they found that 75-100 mg of aspirin used once day was ineffective at avoiding cardiovascular events, sudden cardiac death, or cancer, especially in those who smoked or took enteric-coated forms, suggesting that its dosage is too low for treatment (136). Sulindac was the subject of a further double-blind, placebo-controlled investigation in FAP patients. It was discovered that standard sulindac doses did not prevent adenomas from developing in younger patients with FAP (137) despite the fact that the number of scientific experiments with sulindac was significantly lower and was too small to be trusted. They were either given 75 or 150 mg orally twice a day of sulindac or identically looking placebo tablets for 48 months. Contrarily, celecoxib has a proven track record of protecting patients who have previously experienced sporadic colorectal adenomas from developing the condition again. Over 1,500 patients participated in the PreSAP (prevention of sporadic adenomatous polyps) and APC (adenoma prevention with celecoxib) trails. Both studies findings-one evaluating celecoxib at a daily dose of 400 mg for 3 years and the other evaluating daily doses of 400 and 800 mg are in an agreement with each other. Celecoxib’s effectiveness in preventing adenoma recurrence improves with dosage (138, 139). Celecoxib’s effectiveness in treating various tumor types when administered in conjunction with cystostatic medicines or monoclonal antibodies such as gemcitabine, cisplatin, fluorouracil, or cyclophosphamide has been studied. Moreover, in studies involving certain patient population, rofecoxib has shown to have a lower incidence of adenoma recurrence (58). Usually non aspirin NSAIDs use is associated with increased risk of cardiovascular risk and gastrointestinal bleeding which limit their use in CRC chemoprevention (140). However certain case control studies such as the one based on Danish population analyzing non aspirin NSAID use (average daily dose & gt; or = 0.3) was associated with a substantial reduction in CRC risk. Aspirin and non-selective NSAIDs (SIR 0.74 [0.71-0.77]), but not COX-2i, were linked to lower risk of GI malignancies including CRC, according to a Swedish population-based analysis of persons taking frequent NSAIDs (cumulative exposure of 6 months) (141). Another prospective cohort study analysis found that using non-aspirin NSAIDs was linked to a decreased risk of CRC in postmenopausal women (142).

Chemoprevention necessitates the continuous administration of NSAIDs. The case for prescribing a chemopreventive medication is more convincing when the patient’s CRC risk is higher, and the drug’s cumulative side effects are less severe. Traditional NSAIDs have adverse effects that worsen over time, particularly in older patients who take other drugs due to comorbidities that interact with the chemopreventive agent (107, 143). As a result, the CRC risk must be significantly more than the 5% likelihood that a person at average risk will develop CRC in order to sustain the lifetime treatment of a typical NSAID. The use of NSAIDs for cancer chemoprevention is not advised despite the substantial evidence of activity because of the risk of serious renal, gastrointestinal, and cardiovascular adverse effects that arise from COX inhibition and the suppression of physiologically significant prostaglandin (111, 133). The chemopreventive efficacy of NSAIDs is also insufficient, albeit it is unclear whether this deficiency is brought on by dosage restrictions or resistance mechanisms. Hence preventing NSAIDS from getting into more clinical trials and FDA approval in CRC chemoprevention.

Patients who smoked heavily and had a high BMI had decreased ability to benefit from the chemo-preventive effects of NSAIDs, especially aspirin. Ibuprofen use was linked to a lower incidence of CRC in a different cohort study of patients with germline mismatch repair gene mutations (144, 145). The use of both aspirin and non-aspirin NSAIDs was associated with a reduced risk of cancer, including CRC. The FDA has authorized the use of NSAIDs as analgesics, antipyretics, and anti-inflammatory drugs. These qualities allow NSAIDs to be used to treat a wide range of illnesses, such as migraines, pyrexia, gout, arthritic disorders, muscle pain, and dysmenorrhea, and as an opioid alternative in some cases of severe trauma (145). Colorectal cancers with PIK3CA mutations or COX2 overexpression appear to have a stronger correlation between NSAID and aspirin usage and decreased mortality. Thus lending credence to the idea that NSAIDs might be used as adjuvant therapy for CRC. Optimizing the timing of NSAID use as an adjuvant treatment is clinically important. The synergistic anticancer effect of aspirin, biologically, might be explained by the stimulation of apoptosis through a COX-dependent or COX-independent mechanism (107, 146), the decrease of metastatic risk by preventing the contact between platelets and circulating cancer cells (109, 111, 147, 148), or the modification of the antitumor immune response (112, 149). In the end, NSAIDs may have more than one target and most likely has several adjunctive effects.

Despite the immense potential of NSAIDs as chemo-preventive agents, their use in CRC chemoprevention encounters many challenges. The poor acceptability and cost of screening colonoscopies are the two factors that make chemoprevention of colorectal cancer (CRC) a viable approach. The most promising treatment agents are those NSAIDs, which are presently not advised for the prevention of CRC (150). NSAIDs’ limited chemo-preventive effectiveness is exacerbated by their considerable toxicity, which can be cumulative. These limitations can be curbed by the use of drug combinations, and the development of certain classes of NSAIDs that are chemically modified (for example – phospho-NSAIDs, nitro-NSAIDs, sulindac) and thus have prolonged safety than any other type of NSAIDs like those of conventional ones (150). One of the major challenges for using NSAIDs as a chemo-preventive drug is identifying the subjects who will gain the most from the chemo-preventive medication and those who are at potentially higher risk (151). The development of biomarkers that are predictive and techniques to reliably evaluate risk would be immensely beneficial in this case. Another challenge is optimizing chemo-preventive drug delivery time, dosage, and duration. According to research, very brief durations of agent administration may be necessary and can prevent colon carcinogenesis at an extremely early stage (152). The dosage and duration of NSAID administration might therefore be adjusted to ensure that the least amount of NSAID is utilized for the shortest duration of time. Furthermore, for individuals at risk, starting such an intervention at an early age may be beneficial. Also, the use of other new or combined agents or those agents that prevent other diseases in addition to colorectal cancer has its own merits (153). A meta-analysis of aspirin’s role in preventing CRC and other malignancies in recent years published in May 2009 showed frequent aspirin use is linked to a lower risk of cancer. However, this theory raises various issues, such as the best aspirin dose and the prevention of gastrointestinal bleeding brought on by prolonged aspirin usage. Thus, there is still much debate about the use of aspirin in the prevention and treatment of cancer (154).

NSAIDs are associated with other serious non-cancerous conditions also. As it was recently revealed that using NSAIDs increases the risk of myocardial infarction (155). Among the medications examined were Celecoxib, ibuprofen, diclofenac, naproxen, and rofecoxib (156). According to a study, many NSAID users reported gastrointestinal side effects ranging from nausea, slight pain, and dyspeptic symptoms to serious problems like bleeding, peptic ulcer rupture, and intestinal blockage (157). Peptic ulcer illness in the past, age, and concurrent aspirin usage are all significant risk factors for developing GI side effects in NSAID users (158, 159). NSAIDs are known for having substantial renal side effects, which in extreme situations might result in renal failure, in addition to cardiovascular and gastrointestinal problems (160). A higher risk has been noted in previous research for acute renal failure. Thus, the use of NSAIDs in the treatment and prevention of cancer must be carefully evaluated and there must also be a balance between the risks and the benefits (5).

The number of studies on CRC chemoprevention has grown. Although NSAIDs have shown the most promise, only those with a greater risk of CRC predisposition syndromes, such as Lynch syndrome or FAP, have been advised to take them as chemopreventive medicines (161). The ideal CRC chemoprevention drug is elusive for the majority of patients. Finding new colonic neoplastic pathways that can be targeted as well as developing drug combinations that maximize efficacy and reduce toxicity are obstacles to CRC chemoprevention. It’s crucial to establish if more typical intermediate endpoints, like ACF or adenomas, may be employed given the generally low incidence of CRC in populations at average risk. Identifying subgroups based on genetic characteristics that influence treatment response, a history of polyps, and the subtype of a polyp is vital to determine which subgroups are most likely to benefit from chemoprevention drugs with the lowest degree of risk. CRC chemoprevention research must overcome obstacles including the necessity for funds to finance lengthy trials that enlist lots of participants and the requirement to validate results in various ethnic groups and geographical regions. Since many possible chemoprevention medicines are sold as over-the-counter drugs or dietary supplements, it is crucial to get reliable data on risk since their widespread usage might skew study results. It seems doubtful that CRC screening will ever be replaced as the main form of prevention by chemoprevention. The ability to prove the effectiveness of chemoprevention techniques in clinical trials will become more challenging as screening rates rise and CRC incidence and death decline (162, 163). Therefore, studies in groups who regularly receive CRC screening will need to show a stronger protective impact to significantly support chemoprevention in addition to screening. In conclusion, a chemopreventive drug that is generally effective, safe, affordable, accessible, and simple to use is appropriate for CRC. The promise of lowering CRC risk and lowering its morbidity and mortality makes CRC chemoprevention an activity worth continuing to pursue, even if it is difficult to discover a chemoprevention medication that complies with these requirements.