Vascular endothelial growth factor (VEGF) is the most potent angiogenesis inducer. It stimulates endothelial cell proliferation, migration, differentiation and also vessel branching (reviewed recently in [18]). During the angiogenesis process, VEGF is secreted by several cells including endothelial cells and tumor cells [19].

Some cancer cells express one of the two VEGF receptors, VEGFR-1 or VEGFR-2 (Flk/KDR), and can be stimulated by VEGF signaling [20–22]. VEGF is not only a mitogen but also a survival factor [23] the overexpression of which can induce resistance to chemotherapeutic drugs in soft tissue sarcoma [24]. VEGFR-2 expression was correlated with chemoresistance in non-small-cell lung carcinoma [25] whereas the loss of VEGF expression in colorectal cancer cells caused an increase in apoptosis (spontaneous and chemotherapy induced) [26]. VEGF modifies the apoptotic signaling pathway in endothelial cells by inducing the expression of anti-apoptotic proteins including survivin and Bcl-2 [27–29], and the activation of the PI3K/Akt survival pathway [30]. Bcl-2 expression or phosphorylation can also be up-regulated by VEGF in VEGFR⁺ primary and/or immortalized cancer cells from different sources, leukemia [31,32] or in breast cancer [33]. In chronic lymphocytic leukemia B cells, VEGF interacts directly with STAT1 and STAT3 leading to the up-regulation of other anti-apoptotic proteins, Mcl-1 and XIAP, and protection from cell death [34]. Pericytes, specialized in the stabilization and maturation of vessels, can promote endothelial cell survival [35]. They act on endothelial cells by stimulating the expression of VEGF via NF-κB signaling. By secreting vitronectin, the pericytes induce the activation of endothelial cell integrin α_V leading to the increased expression of the anti-apoptotic protein Bcl-w [36]. Interestingly, such prosurvival signaling induced by VEGF can be converted into pro-cell death signals by TGFβ1, a regulator of tissue morphogenesis, during an apoptosis step required for angiogenesis [37].

Chemoresistance acquisition and angiogenesis are two linked processes. Indeed one study showed that chemoresistance acquisition by continuous treatment in neuroblastoma cells was related to transcriptome modifications in angiogenic genes and correlated with a positive influence on angiogenesis in vitro and in vivo [38]. In breast cancer cell lines, hypoxia-stimulated VEGF expression was increased by overexpression of Bcl-2 [39]. In contrast, down-regulation of anti-apoptotic proteins Bcl-2 or survivin by different approaches improved the sensitivity to treatment (radiotherapy or chemotherapy) and inhibited VEGF expression and angiogenesis in two different tumor xenograft models (prostate and colon) [40,41].

In addition to the tumor cells, endothelial cells also need to be eradicated by any cancer treatment to minimize the risk of recurrence. These cells develop their own specific apoptosis resistance pathways depending on the model studied and death inducer. FGF-2, which is also an angiogenic factor, promotes apoptosis resistance in endothelial cells after radiation treatment [42] or growth factor deprivation [43]. It has the same prosurvival effects as VEGF, upregulating Bcl-2 and survivin expression [44] and activating the protein kinase Akt [45,46].

Both FGF-2 and VEGF protect endothelial cells from apoptosis after exposure to different chemotherapies, and have an additive effect due to activation of PI3K/Akt signaling pathway [26]. Cancer cells can act on endothelial cells and protect them from apoptosis after radiation through their secretion of VEGF and the subsequent activation of pro-survival gene expression [47]. Moreover, in different cancer models, tumor-associated endothelial cells have been shown to differ from those derived from normal organs or cells. For example, the tumor-associated endothelial cells showed increased chemoresistance compared to normal endothelial cells through the expression of PAX2 in renal carcinoma [48], the overexpression of survivin in a glioma model [49], and the activation of an NF-κB dependent pathway promoting Akt and VEGF expression and cell survival in hepatocellular carcinoma [50]. These molecular pathways are also associated with the stimulation of angiogenesis [48–50].

Specific anti-angiogenic drugs have been developed over the last few years most of which target VEGF signaling (VEGF or VEGFR).

Anti-angiogenic therapies used as a single agent have been shown since the first preclinical studies to inhibit angiogenesis and diminish tumor growth [51] and also permit an increase in tumor cell apoptosis [52]. They are used in combination with conventional therapies for their ability to improve delivery [53] by reducing interstitial fluid pressure [54]. Moreover, the use of two angiogenic inhibitors together has shown very promising results in a glioma model where a VEGFR2 inhibitor alone did not permit blood vessel regression. A combination with an inhibitor of PDGFR-β overcame the survival mechanism by targeting pericytes, mediators of endothelial cell survival mechanisms, thus demonstrating that in blood vessels, resistant processes work cooperatively [55]. In clinical trials, anti-angiogenic therapies have been shown to improve the response to chemotherapy in different types of cancer [56,57] however when administered alone, they do not permit an improvement of long term benefit [58].

The paradox between anti-angiogenic therapy expectations and clinical observations is explained by the recent concept developing the idea that anti-angiogenic treatments provide the best results when they provoke “vessel normalization”. These drugs do not abrogate tumor angiogenesis but rather turn transiently anarchic tumor blood vessels, caused by overexpression of angiogenic factors, into normal ones. Not only does this allow a better delivery of chemotherapy and increase the sensitivity to radiotherapy but it also decreases tumor cell extravasation and migration in the blood circulation by reestablishing the endothelial cell barrier [55,59]. As with every therapy, resistance to an anti-angiogenic drug can occur mostly due to its mechanism of action. Indeed their aim is to suppress angiogenesis causing hypoxia [60].

To survive, tumor cells develop several adaptive mechanisms including metabolic shift (oxidative metabolism to glycolysis) and apoptosis resistance [61]. The microenvironment can positively influence tumor apoptosis resistance via the activation of resistance signaling pathways. Extreme conditions imposed by the microenvironment can also influence cancer cells by obliging them to modify their phenotype in order to overcome the hypoxia and survive. One of the major ways for tumors to survive in hypoxic conditions is to escape from the local area, thus explaining the high migration and invasion potential of these cells [60]. Hypoxia is drastic for cancer cells but leads to genetic instability and the selection of the most malignant cells with the highest metastatic abilities [62]. The key molecule acting during hypoxia is hypoxia-inducible transcription factor 1 (HIF-1) which transactivates hundreds of genes including angiogenic and autocrine growth factors and receptors, glycolytic enzymes and extracellular proteases [63]. Hypoxia particularly induces VEGF expression in tumor cells thereby activating anti-apoptotic pathways described in point 3.3. [64]. Hypoxia can also induce an inflammatory state through HIF1 and NF-κB activation leading to the secretion of chemokines and cytokines able to recruit inflammatory cells which also release VEGF [65].

Anti-angiogenic therapies, through the hypoxic response they cause in cancer cells, could be responsible for an enhancement of metastasis and invasion [66] and may bring about a more aggressive behavior. This point is controversial and still under investigation. Indeed, while some preclinical studies have shown that local invasiveness and metastasis are triggered by anti-VEGF treatments [67,68] others found no effect on metastasis [69,70].

In the particular case of multiple myeloma (MM), a plasma cell cancer, the adhesion between MM cells and bone marrow fibroblasts leads to the secretion of IL-6 by the latter [71]. This pleiotropic cytokine has demonstrated the capacity to induce the resistance of MM cells to apoptotic stimuli and chemotherapeutic drugs via the Jak/STAT pathway and the expression of the anti-apoptotic protein Bcl-xL [72].

Myofibroblasts or carcinoma-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment. While their tumor-promoting effects are well known, their origin and a clearly characterized phenotype have not been well established. They can be distinguished from normal fibroblasts by their expression of certain markers like alpha smooth muscle actin (αSMA), fibroblast activation protein (FAP), tenascin-C or desmin [73].

Both cancer cells and CAFs are able to secrete prostaglandin E₂ (generated by COX-2 activation) and sphingosine-1-phosphate (S1P), that can act in an autocrine or paracrine fashion to mediate cell survival and chemoresistance via PI3K-Akt/PKB pathway activation [74]. In cholangiocarcinoma, CAFs also secrete platelet growth factor BB (PDGF-BB) which protects the cholangiocarcinoma cells from TRAIL cytotoxicity thus implying involvement of the Hedgehog (Hh) pathway [75].

CAFs can regulate extracellular matrix composition by secreting periostin, a ligand of αvβ3 and αvβ5 integrins, which allows cancer cell adhesion and migration but also apoptosis resistance by PI3K-Akt/PKB activation in breast cancer models [76]. Thanks to their expression of the serine protease FAP, CAFs allow collagen I cleavage and thus extracellular matrix remodeling [77]. By interacting with collagen fibers in an integrin-dependent manner, these cells exert an increased tension between the fibers and ultimately increase the interstitial pressure which diminishes drug uptake and efficacy [78]. Concordantly, fibroblast-derived 3D matrix has been shown to promote resistance of the PANC-1 line (pancreatic cancer cells) to taxol [79].

Myofibroblasts have been shown to enhance chemoresistance in a pancreatic carcinoma model via an epigenetic inhibition of STAT1 and a reduced expression of caspases (8, 9, 7 and 3). They achieve this by inducing the expression of DNA methyltransferase 1 (DNMT1) and CpG DNA-hypermethylation [80].

Their abundance in the tumor stroma and various effects on tumor progression and apoptosis resistance has made CAFs a new target in anticancer therapy. The desire to target CAFs instead of normal fibroblasts drove the development of, among others, anti-tenascin-C or anti-FAP molecules. Results from a phase II trial of the anti-tenascin monoclonal antibody 81C6 followed by chemotherapy in malignant glioma were promising compared to the control group [81].

Stromal fibroblasts are genetically stable compared to tumor cells and constitute a reliable target for immunotherapy. Loeffler et al. developed a DNA vaccine which targets FAP by activating CD8⁺ T cells in order to specifically kill the CAFs and thereby decrease collagen I expression and enhance drug uptake. The combination of such an anti-FAP vaccine with doxorubicin brought about an inhibition of tumor growth and complete tumor rejection in half of the tested mice whereas there was no survival with doxorubicin treatment alone [82].

MSCs could promote such chemoresistance by direct cell-cell contact. Firstly Xu et al. proposed that TGF-β1 produced by BM stromal cells promotes the survival and chemoresistance of leukemia cells via direct cell-to-cell interactions [87]. They showed that the blockade of TGF-β signaling by LY2109761, which effectively inhibited the pro-survival signaling, could enhance the efficacy of chemotherapy against myelo-monocytic leukemic cells in the BM microenvironment. Rafii et al. demonstrated the capacity of CA-MSCs (called Hospicells in their study) to confer chemoresistance to ovarian and breast cancer cells by direct cell-cell contact and the exchange of membrane patches and MDR proteins (oncologic trogocytosis) [5,7].

Jin et al. showed that MSC cocultured with KBM-5 leukemia cells protected the latter from imatinib-induced cell death. As these anti-apoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by the inhibitor of integrin-linked kinase QLT0267, they suggested that the upregulation of CXCR4 by imatinib promotes migration of chronic myelogenous leukemia (CML) cells to bone marrow stroma, causing G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells [89].

Roodhart, Hao and Castells showed that MSCs could induce chemoresistance through the release of factors in the neighbourhood of tumors. Castells et al. showed that supernatants of CA-MSCs as well as BM-MSCs were able to induce development of chemoresistance by reducing apoptosis. They observed that CA-MSC secretions were able to confer carboplatin resistance to ovarian cancer cells by inhibiting the activation of effector caspases and apoptosis blockade. The activation of PI3K/Akt pathway signaling and phosphorylation of the downstream target Xiap underlined their implication in ovarian cancer chemoresistance.

The factors released in the tumor microenvironment have yet to be identified. Using activated MSCs, Roodhart et al. also showed that MSCs are potent mediators of resistance to chemotherapy and revealed targets to enhance chemotherapy efficacy in patients. They claimed that MSCs per se do not induce chemoresistance, rather endogenous MSCs become activated during treatment with platinum analogs and then secrete factors that protect tumor cells against a range of chemotherapeutic drugs. Through a metabolomic approach, they identified two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), that in minute quantities induce resistance to a broad spectrum of chemotherapeutic agents. Blocking central enzymes involved in the production of these PIFAs (cyclooxygenase-1 and thromboxane synthase) prevents such MSC-induced resistance [3]. Hao et al. demonstrated in myeloma that the secretion of IL-6, VEGF and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, they associated the chemoresistance to the suppression of miRNA-15a expression by the BM-MSCs [88].

Pasquet et al. showed that CA-MSCs could induce the formation of hypoxic niches around tumors associated with a high expression of HIF1-α [84]. Benito et al. indicated that interactions between leukemia cells and the BM microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs [90]. They correlated the hypoxic areas within the BM microenvironment with a survival advantage to hematopoietic cells and showed that hypoxia promotes chemoresistance in various ALL derived cell lines.

In their study, Teng et al. have shown that DNA hypermethylation within a specific tumor suppressor gene is sufficient to fully transform a somatic mesenchymal stem cell. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Moreover, the cells retained sensitivity to neuron- and osteocyte-induction and displayed both lineage-specific markers and stem cell markers in xenografts. Teng et al. proposed that under the influence of different environmental niches, these transformed stem cells could give rise to tissue-specific cancers [10].

Monocytes and macrophages derive from myeloid cells, which are located in the bone marrow. After maturation, monocytes circulate in the bloodstream and can migrate into tissues where they differentiate into macrophages. Depending on the environmental context and the tumor development stage, activated macrophages can be separated into two distinct phenotypes: M1 (classical activated), which inhibit tumor growth and M2 (alternative activated), which are pro-tumoral. While the exact definition is still controversial, it is clear that tumor-associated macrophages (TAMs) consistently present a highly immunosuppressive M2 profile [95].

Within developed tumors, there is a balance in favor of tumor progression and the chemoprotective effects of TAMs being closely associated with reduced cytotoxic CD8⁺ T-lymphocyte activity. Indeed, hypoxic TAMs suppress CD8⁺ T-lymphocytes activity by the activation of inducible Nitric Oxyde Synthase (iNOS) and arginase I (ArgI, liver-type) in a HIF1α-dependant manner. Moreover, the inhibition of TAM recruitment by a CSF1 neutralizing antibody in mammary tumors leads to a better chemosensitivity to paclitaxel, reduced tumor progression and metastasis that was associated with an increased survival of CD8⁺ T-cells. [96].

Shree et al. showed that cathepsin-expressing macrophages protect breast cancer cells from cell death induced by the following chemotherapeutic drugs: taxol, etoposide and doxorubicin. They highlighted the growing importance of combining anti-microenvironment drugs with classic chemotherapy. Indeed, the combination of anti-cathepsin with taxol treatment enhances the anti-tumor efficacy, the late-stage survival and decreases the metastatic burden compared to taxol alone in a breast cancer mouse model [97]. Mononuclear cells (monocyte-like cell line U937) prevent pancreatic cancer cells from camptothecin and genistein induced apoptosis in vitro by interleukin-1β-mediated expression of cycloxygenase-2 (COX-2) and the production of prostaglandins [98].

While TAMs are pro-tumoral and enhance chemoresistance, the presence of cytotoxic CD8⁺ lymphocytes (CTLs) is usually associated with a good prognosis and a better response to chemotherapy. Indeed, CTLs have an anti-tumor activity in secreting perforine or granzyme which induce apoptosis in tumoral cells. A high density of CD8⁺ cells has been correlated with a better response to chemotherapies in primary colorectal cancer or liver metastases [99,100]. Denkert et al. showed that the presence of lymphocytes infiltrates in breast cancer is correlated with an increased efficacy of anthracycline and taxane neoadjuvent chemotherapy [101] (Figure 1, Figure 3(A,B)).

Interactions between chemotherapy and CTLs are very tight and the efficacy of the latter can be increased after a first treatment. Indeed, a cyclophosphamide treatment in a mouse model of mesothelioma sensitizes tumor cells to the TRAIL-mediated death induced by CTLs [102]. Moreover, a chemotherapy treatment (based on paclitaxel, cisplatin or doxorubicin) leads to an up-regulation of mannose-6-phosphate receptors which increases the permeability of tumor cells to granzyme B released by CTLs [103]. Mattarollo et al. showed, in a mouse model of breast adenocarcinoma, that a doxorubicin treatment enhances the proliferation of IFN-γand IL-17 producing CD8⁺ T cells. In return, they demonstrated a positive correlation between the CD8α, CD8β and IFN-γ expressionb and the efficacy of doxorubicin treatment in patients with breast cancer [104]. Chemotherapy and CTLs thus present a synergical effect. The association between a classical chemotherapy treatment and immunotherapy present a very interesting way of therapeutic development.

γδ T lymphocytes are CD3⁺ cells expressing a TCR with γ and δ chains. They represent a small population of T lympochytes (5%) and show anti-tumor properties. Recently, the team of Zitvogel demonstrated that IL-17-producing γδ T lymphocytes were implied in the efficacy of chemotherapy. Indeed, in a mouse model lacking these γδ T cells, there is no IL-17 production and CTLs fail to invade tumor. The chemotherapy efficacy is thus impaired [105].

Regulatory T cells, a Fox3p⁺ subpopulation of CD4⁺ T lymphocytes, are also implied in response to chemotherapy with controversial conclusions. Their presence is associated with a better survival exclusively in chemotherapy treated patients with an early breast cancer [106]. However, some other studies shown that the recruitment of Tregs in breast cancer can be associated with a bad clinical outcome and a higher risk of relapse [107,108]. In a recent work, Liu et al. demonstrated that the equilibrium between CTLs and Tregs in tumors is primordial to predict their response to chemotherapies. In an advanced non-small cell lung cancer context, a high Fox3p⁺/CD8⁺ ratio is associated with a poor response to platinum-based chemotherapy [106].

CD4⁺ naïve T lymphocytes can, after the activation by antigen-presenting cells (APCs), differentiate into effector cells: T helpers (Th). The interactions with APCs and the nature of cytokine environment will define the subtype obtained: Th1, Th2 and Th17. These cells are implied in the activation of the immune system but differ in their cytokine production and biological functions.

Th1 cells are characterized by IFN-γ and TNF-α secretion and are responsible for the activation of CTLs or anti-tumoral macrophages. Tosilini et al. recently showed that the presence of Th1 cells was correlated with a prolonged disease-free survival in patients with colorectal cancer [109].

On the contrary, Th2 cells secrete IL-4 and enhance tumor progression in activating, for example, tumor-associated macrophages [110]. A few data directly links Th1 and Th2 cells with chemotherapy efficacy. However, the production of IL-4 (Th2 cytokine) is associated with resistance to chemotherapy and thyroid cancer [111].

Th17 secrete the pro-inflammatory cytokine IL-17 and present both anti-tumor and pro-tumor activities. The microenvironmental context is highly important in the interactions between Th17 cells and the tumor. However, many data link the IL-17 production, as previously cited for γδ lymphocytes, with the establishment of CTLs in the tumor bed and thus the anticancer efficacy [112].

As reviewed here and by Ding and Zhou, targeting CD4⁺ T cells constitutes a new way of immunotherapy in order to increase the chemotherapy efficacy [113].

Among stromal cells of immune origin, mast cells (MCs) have been observed to infiltrate tumor masses. Indeed in lymphoid malignancies infiltrating MCs have been demonstrated to support clone survival and proliferation, and to negatively affect prognosis [114]. Mechanisms of tumor promotion involve the release of proangiogenic factor such as FGF-2 [115] or VEGF and matrix metalloproteinases (MMP9) but also of immunosuppressive cytokines like interleukin-10 [116]. As described before those molecules are also implicated in chemoresistance acquisition by tumor cells and MCs could be potential targets for chemotherapy treatment. They have been shown to be a prototypical off-target cell for imatinib therapy in cancer, due to their constitutively high expression of c-Kit, strong reliance on c-Kit signal for development and activation, and aforementioned tumor promoting activities [114].