Today, the critical role of stroma in the carcinogenic process is clear. Fibroblasts are the main cellular component of tumor stroma (Cancer-Associated Fibroblasts, CAF). This is a heterogeneous cell population of multiple origins (tissue-resident fibroblasts, myeloid precursors, pericytes and adipocytes, among others) and features that is acquired via the change to an “activation phenotype”. It is thought to promote cancer invasion, angiogenesis and metastasis; inhibit the immune response; and reduce intratumoral delivery and the activity of chemotherapeutic agents [244,245]. However, the protective effects of CAF have also been described in some systems, and reprogramming their phenotype is accepted as a more advisable strategy than their elimination [246,247]. Early studies showed that VDR agonists have antifibrotic and antitumoral effects by antagonizing TGF-β in the intestine, liver, and pancreas [248,249,250,251,252].

1,25-(OH)₂D₃ regulated over one hundred genes in human CAF isolated from tumor biopsies of five breast cancer patients [253]. The induced gene signature reflects an antiproliferative and anti-inflammatory effect of 1,25(OH)₂D₃. Importantly, 1,25-(OH)₂D₃ inhibits the protumoral action of human colon CAF by reprograming them to a less activated phenotype. Thus, 1,25(OH)₂D₃ reduces the capacity of CAF to alter the ECM and their ability to promote the migration of colon carcinoma cells [254]. 1,25-(OH)₂D₃ regulates over one thousand genes in colon CAF that are involved in cell adhesion, differentiation and migration, tissue remodeling, blood vessel development, and the inflammatory response. Remarkably, 1,25(OH)₂D₃ imposes a gene signature that correlates with a better prognosis for colon cancer patients [254]. Curiously, in contrast to the antagonism reported in colon carcinoma cells, 1,25-(OH)₂D₃ and Wnt3A have an additive, partially overlapping effect in colon fibroblasts [255,256]. In line with the results in colon CAF, 1,25(OH)₂D₃ decreases the amount of miR-10a-5p found in the exosomes secreted by human pancreatic CAF, which attenuates the promigratory and pro-invasive effects that these CAF exert on pancreatic carcinoma cells [257]. Of note, a recent study reported that calcipotriol promotes an antitumorigenic phenotype of pancreatic CAF by reducing the release of prostaglandin (PG) E₂, IL-6, periostin, and other factors. However, it reduces T-cell-mediated immunity [258]. Clearly, the action of VDR agonists on fibroblasts associated with distinct human cancers is a highly interesting, open line of research.

Cancer stem cells (CSC) are supposedly a small population of cells present in tumors that are responsible for tumor initiation, growth, malignization, metastasis, and resistance to therapies. They originate from the mutational and epigenetic alteration of normal stem cells that maintain the homeostasis of tissues in adult life and behave as a source of new functional differentiated cells following injuries or in aging. The characterization and study of CSC present two unresolved problems: (a) the lack of confirmed universal or even tissue-specific markers, and (b) the existence of cell plasticity in tumors that implies differentiation/dedifferentiation processes during tumorigenesis and thus the lack of a stable stem phenotype but, instead, interconversion of stem and non-stem cells.

At present, there are two systems to study CSC: organoid cultures generated by CSC present in patient-derived tumor biopsies and subcultures of established, immortal tumor cell lines enriched in populations of cells expressing putative CSC markers and/or selected by their capacity to grow in suspension. Clearly, fresh, primary organoids are a more valuable system. They are three-dimensional (3D), self-organized multicellular structures generated by normal stem cells or CSC (that allow matched normal and tumor organoids to be obtained from a patient) that grow embedded in an ECM covered by a complex, tissue-specific, usually serum-free medium [259,260]. Organoids recapitulate some of the features of a particular organ or tumor of origin and are quite stable genetically, and thus are considered a better system to study cancer processes than 2D cell lines grown for decades on plastic dishes [261]. 1,25(OH)₂D₃ profoundly and differentially regulates the gene expression profile of colon cancer patient-derived normal and tumor organoid cultures. 1,25(OH)₂D₃ induced stemness-related genes (LGR5, SMOC2, LRIG1, and others) in normal but not tumor organoids [262]. In both normal and tumor organoids, 1,25(OH)₂D₃ reduced cell proliferation and the expression of proliferation and tumorigenesis genes that affected only a few Wnt/β-catenin target genes (MYC, DKK4). Importantly, 1,25(OH)₂D₃ induced some features of epithelial differentiation in tumor organoids cultured in proliferation medium, such as microvilli, adhesion structures, partial chromatin condensation, and increased cytoplasmic organelles. These effects were also observed in rectal tumor organoids [263].

Concordantly, 1,25(OH)₂D₃-regulated genes were involved in cell proliferation, differentiation, adhesion, and migration in another study using patient-derived colon organoids [264]. Moreover, MDL-811, an allosteric activator of the sirtuin (SIRT)6 deacetylase, reduced cell proliferation in colon carcinoma cell lines and patient-derived organoids and has a synergistic antitumoral effect in combination with vitamin D in Apc^min/+ mice [265]. However, conflicting data have been found in normal, nontumoral organoids: whereas 1,25-(OH)₂D₃ increased the stemness genes and the undifferentiated associated cell phenotype in organoids from healthy colon and rectum tissues of a dozen individuals [262,263], it enhanced the differentiation of organoids established from a benign region of a radical prostatectomy from a single patient [266].

A series of studies have examined the action of VDR agonists on putative breast cancer stem or progenitor cells identified by some markers (CD44^hi/CD24^low and/or ADH1⁺) that can grow as floating, nonadherent spheres (mammospheres). In these systems, 1,25(OH)₂D₃ or the BXL1024 analogue reduced the population of putative CSC and the formation of mammospheres and the expression of pluripotency markers (OCT4, KL-4), Notch ligands and target genes, and genes involved in proliferation, EMT, invasion, metastasis, and chemoresistance 32,467,291 [267,268,269].

Organoids formed by cells isolated from patient-derived xenografts (not obtained directly from human biopsies but on injection and growth in mice) that acquired resistance in vitro to Trastuzumab-emtansine (T-DM1; composed of the humanized monoclonal anti-HER2 antibody Trastuzumab covalently linked to the microtubule-inhibitory agent DMI) constitute an intermediate system to the two discussed above. In this system, two vitamin D analogues (UVB1 and EM1) reduce the formation and growth of organoids [270].

1,25-(OH)₂D₃ is an important modulator of the immune system, as reflected by the expression of VDR by almost all types of immune cells [271,272,273]. 1,25-(OH)₂D₃ is an enhancer of innate immune reactions against infections and tumor cells by activating the responsive cells (macrophages, natural killer (NK) cells, and neutrophils). Conversely, and in line with its accepted anti-inflammatory action (that may contribute to the inhibition of cancers associated with chronic inflammation), 1,25-(OH)₂D₃ is commonly presented as a repressor of the adaptive immune reactions by deactivating antigen-presenting cells (induction of tolerogenic dendritic cells) and CD4⁺ type-1 helper T (Th1) response (production of interferon-γ, IL-1, IL-6, IL-12...), and by promoting the suppressive Th2 and Treg responses (production of IL-10, IL-4, IL-5, IL-13...) [273,274]. Moreover, in macrophages, 1,25-(OH)₂D₃ has been proposed to promote a switch from the pro-inflammatory M1 phenotype (producing IL-1β, IL-6, TNF-α, RANKL, COX) towards the anti-inflammatory protumoral M2 phenotype and to reduce the T-cell stimulatory capacity of macrophages [275,276]. This is somehow counterintuitive as it would represent a potential protumoral effect that cannot be easily attributed to a conserved evolutionary agent such as vitamin D. Some other studies discussed below have introduced putative explanations.

Since naïve T-cells express VDR at a very low level that increases only after activation of the T-cell receptor [277], the role of 1,25-(OH)₂D₃ may conceivably be related to the late downregulation of the activated adaptive response. This view agrees with the usual description of repressive 1,25-(OH)₂D₃ action in experimental settings following overstimulation of the cells, and it may constitute a safety mechanism to prevent undesirable long-lasting immune activation, potentially leading to inflammation or autoimmunity [278,279]. Concordant with this idea and the anticancer action of 1,25-(OH)₂D₃, a series of studies have revealed antitumor effects at the level of several types of immune cells.

Interestingly, a study in mice orthotopically implanted with breast tumors has revealed that vitamin D decreases tumor growth and increases the amount of tumor-infiltrating cytolytic CD8+ T-cells, a usual marker of antitumor response. This effect is lost in high-fat diet conditions [280]. Moreover, in pancreatic cancer, 1,25-(OH)₂D₃ inhibits the T-cell suppressive function of myeloid-derived suppressor cells [281].

An important mechanism of 1,25-(OH)₂D₃ is the inhibition of the NF-κB pathway. In turn, this causes the downregulation of multiple cytokines and their effects [282]. 1,25-(OH)₂D₃ inhibits NF–κB at different levels: by inactivating the p65 subunit of the NF-κB complex and upregulating the inhibitor subunit IκB. In addition, 1,25-(OH)₂D₃ inhibits the PG-endoperoxide synthase (PTGS-2, also known as COX-2) [283,284,285]. 1,25(OH)₂D₃ reduces the protumorigenic effect of PG E₂ in prostate cancer cells by inhibiting COX-2 and so decreasing the levels of PG E₂ and two PG receptors (EP2 and FP) [286]. Importantly, vitamin D and calcium favorably modulate the balance of expression of COX-2 and 15-hydroxyPG dehydrogenase, its physiological antagonist, in the normal-appearing colorectal mucosa of patients with colorectal adenoma [287]. Vitamin D enhances the tumoricidal activity of NK cells and macrophages [288,289]. 1,25-(OH)₂D₃ probably has a dual effect of stimulating the differentiation from monocytes to macrophages and their cell killing activity, including antibody-dependent cell cytotoxicity (ADCC). It may later balance these effects by promoting the M1 to M2 phenotypic switch ([279] and references therein). In addition, 1,25-(OH)₂D₃ enhances the susceptibility of hematological and solid cancer cells to NK cell cytotoxicity through downregulation of miR-302c and miR-520c [289].

The potentiation of ADCC of macrophages and NK cells may be a relevant antitumor action of 1,25-(OH)₂D₃ in clinical cases, particularly in patients treated with antibodies, of which the major mechanism of action is ADCC. Thus, several studies have shown that vitamin D deficiency impairs the macrophage and/or NK cell-mediated cytotoxicity of Rituximab (anti-CD20) in diffuse large B-cell, follicular, and Burkitt lymphoma patients [288,290,291], and of Cetuximab (anti-EGFR) in colon cancer cell lines [292]. In addition, some evidence of benefit has been observed in breast cancer patients treated with Trastuzumab (anti-HER2) and in melanoma patients treated with Bevacizumab (anti-VEGF) [290,293].

Agents that target programmed death (PD)-1 or its ligand PD-L1 immune checkpoint inhibitors (ICI) have attracted great attention in cancer therapy. Interestingly, 1,25-(OH)₂D₃ upregulates PD-L1 in human (but not mouse)-cultured epithelial and immune cells [294], while vitamin D treatment increases PD-1 expression in CD24⁺CD25^+int T-cells in Crohn’s disease patients [295] and PD-L1 in epithelial and immune cells in melanoma patients [296]. These data suggest the possibility of combined treatments with VDR agonists and these ICIs, and perhaps others in development.

In conclusion, it is conceivable that 1,25-(OH)₂D₃ works as a general homeostatic regulator of the immune system, ensuring an appropriate global defense against challenges like tumors and infections.

Many studies on animal diet, chemical, genetic, and xenograft models (mainly for colon and breast cancer) have shown the antitumor actions of vitamin D compounds. This in vivo action is difficult to dissect and probably results from a variable combination of mechanisms in the distinct systems that were assayed, including the inhibition of tumor cell growth, EMT, invasiveness, angiogenesis, and metastasis. Importantly, as occurs in cultured cancer cells, vitamin D antitumor action is mostly independent of TP53 gene status [119,187].

The vast array of effects that 1,25-(OH)₂D₃ has in a wide variety of experimental systems of a high number of cancer types agrees with a selected evolutionary role in protection against tumoral processes. The underlying mechanisms include the control of tumor cell survival (autophagy, apoptosis) and phenotype (differentiation), and the inhibition of their proliferation, invasiveness, and metastasis; attenuation of the proliferation and phenotypic features of some CSC; modulation of the physiology of diverse non-tumoral stromal cells (fibroblasts, endothelial cells); and the regulation of several types of immune cells and responses.

Together, these effects reflect a multilevel anticancer action of vitamin D. Therefore, an appropriate vitamin D status of the organism should be maintained to minimize the risk and severe consequences of many neoplasias. Further supporting this, the toxicity of vitamin D supplementation is limited, acceptable, and clearly lower than that of current anticancer drugs and therapies. We are not aware of any other natural or synthetic compound that has such an array of antitumor activities combined with low toxicity. Doubtless, the available experimental results meet Koch’s postulate for biological causality regarding the existence of a global mechanism of action behind the association between vitamin D deficiency and high incidence and, especially, the mortality of several major cancer types found in observational and epidemiological studies. Hopefully, the further development of current and possibly, novel studies on the wide range of mechanisms of VDR agonists in a variety of biological systems will allow us to elucidate the anticancer action of vitamin D (Figure 3).