First-line treatment in patients with CRC is FOLFOX, which includes 5-fluorouracil (5-FU), oxaliplatin, and leucovorin. However, most patients develop resistance to this treatment and die within 1–10 years after its initiation [
57]. Angiogenesis is required for invasive tumor growth and metastasis, which is mediated through VEGF and EGFR. Patients with metastatic CRC are currently treated with irinotecan and immunotherapy (bevacizumab, ramucirumab, and Ziv-aflibercept against VEGF and either cetuximab or panitumumab against EGFR) [
58] DJ-1 (PARK7/CAP1/RS) is a multifunctional protein that protects neurons from oxidative stress by activating Akt/mTOR, MEK/ERK, NF-κB, and HIFα signaling pathways. Overexpression of DJ-1 in many tumor types correlated with promoting cancer cell survival, proliferation, and metastasis. Results recently suggest that DJ-1 is a potential prognostic and therapeutic target in invasive CRC [
59]. More recently, the DART protein MGD007 was designed to co-engage T lymphocytes with CRC cells through the cell surface antigens, CD3 and gpA33, respectively, in order to promote T-cell recruitment and anti-tumor activity [
60]. In addition, novel 89Zr-labeled anti-LGR5 mAbs were developed for evaluating the imaging potential of the CSC marker and were useful for stratifying patients that would respond best to an LGR5-targeted ADC therapy, and for monitoring treatment response in CRC [
61]. Targeting strategies in self-renewal pathways in CSCs, including their pharmacological antagonists Hh ligand Inhibitors (PTCH1 inhibitor or RU-SKI [
62]. GLI Antagonists (TAK-441-trial advanced CRC), SMO Inhibitors, Anti-DLL4/NOTCH Antibodies [
63,
64]. (OMP-21M18, REGN421, and MEDI0639 for anti-angiogenesis), γ-secretase inhibitors [
65] (PF-03084014 inhibitor is generally safe and well tolerated by oral administration in advanced cancer). Wnt ligand inhibitors such as OMP-54F28 [
66] which is a recombinant protein formed by the fusion of the immunoglobulin Fc to the CRD of FZD8 for blocked WNT, are undergoing clinical trials [
67] despite being a promising strategy, it still has limitations such as the systemic toxicity of the antibodies used to block any of the pathways involved in the maintenance of CSC.CRISPR/Cas9 has become a powerful tool for changing the genome of many organisms. The open-label phase I study (NCT02793856) using CRISPR for cancer therapy was programmed cell death protein-1 (PD-1) knockout engineered. PD-1, a member of the CD28 superfamily of T-cell regulators expressed in a wide range of immune cells, including peripherally activated T cells, B cells, monocytes, NK cells, and DCs that consist of an Ig-V like extracellular domain, a transmembrane domain, and a cytoplasmic domain that harbors two tyrosine-based signaling motifs, interacts with two ligands [
68]. These ligands, PD-L1 (CD274 or B7H1) and PD-L2 (CD273), were found expressed in some tumor cells. PD-L1 is expressed in many cell types such as vascular endothelium, reticular fibroblasts, non-mesenchymal stem cells, islet cells, astrocytes, neuronal cells, and keratinocytes. Interactions between the extracellular domains of PD-L1 and PD-1 attenuate T cell-activating signals and lead to inhibiting proliferation, survival, and production of growth factors such as EGF, TGF-β, and GM-CSF, and cytokines such as INFγ, TNF-α, IL-6 and IL-17. Activation of the PD-1/PD-L1 signaling pathway causes immunosuppression of T cell function, which is considered the main factor responsible for response immune escape [
69]. However, cancer stromal cells can contribute to tumor microenvironment upregulates PD-L1 expression, by express GM-CSF and VEGF and promotes immune suppression. This effect is called “adaptive immune resistance”, because the tumor protects itself by inducing PD-L1 in response to IFN-γ produced by activated T cells. T cells ex vivo are evaluated for treating metastatic non-small cell lung cancer that has progressed after all standard treatments. Patients enrolled in the gene-editing trial provided peripheral blood lymphocytes and PD-1 knockout of T-cells by CRISPR/Cas9 performed ex vivo. The edited lymphocytes were selected, expanded and subsequently infused back into the patients. Four other trials applying the same concept of PD-1 knockout for treatment have been registred for other cancer types, including prostate, bladder, esophageal and renal cell cancer [
62]. Recent studies propose as a target for colorectal cancer EGFR (overexpressed in 60–80% of aggressive tumors) or CAE as chimeric antigen receptors allow T-cells to recognize tumor cells and quickly destroy them [
70]. This strategy is novel with safe and efficient results; mainly in hematological tumors with a lower response in solid tumors. New treatment approaches are still required since these present disadvantages such as side effects after their administration. In addition, it is still necessary to evaluate for prolonged periods if the resident tumor cells that do not evade this treatment by EMT are not able to develop metastasis. A recent report demonstrated that PD-L1 induces ZEB1, which activates OCT4 and Nanog signaling and upregulation of EMT on CSC. These promote chemoresistance and metastasis by increased phosphorylation of AKT and ERK, resulting in activation of the PI3K/AKT and MAPK/ERK pathways and an increase of MDR1 expression. Recently, Nivolumab, an anti-PD-L1 drug was approved for metastatic CRC resistant to fluoropyrimidine, oxaliplatin and irinotecan [
68,
71]. This is because the therapeutic targets used are not specific to this cell population (CSC) and the pericytes, as the cells required to ensure the establishment of the metastases have not yet been taken into account.
Circular RNAs (circRNAs) are abundant and important members of the non-coding RNA family which are generally expressed at low levels and exhibit cell-type-specific and tissue-specific patterns, with an average half-life of 19–24 h and whose function remain mostly unknown [
72]. There has recently been considerable attention on circRNA as a molecule that regulates or controls miRNA expression; therfore, they play a significant role in many fields of cancer biology. In tumor biology, circRNA emerges as an effective biomarker for the detection of cancers mainly because it allows differenciation between a normal cell and a tumor cell as well as exhibiting dynamic global changes in its expression levels during tumor progression [
73]. In addition, because circRNA have normally been detected in saliva and blood, they can help as biomarkers that are able to predict sensitivity, the risk of metastasis or the prognosis of treatment. An example as a predictor of 5FU resistance, Xiong et al. identified three upregulated circRNAs (0007031, hsa_circ_0000504 and hsa_circ_0007006) in CRC by microarray analysis [
74,
75]. However, until now they have not been used for therapeutic purposes. Also, the importance that these could have in colon cancer is unknown.