Cancer is one of the leading causes of death worldwide with nearly 2 million new cases in 2022 (1). It is accounted for an approximately 10 million deaths in 2020 (2) and lung cancer alone contributed to 350 deaths per day in 2022 (1). This demands the development of sustainable treatment options to cure and manage cancer. Cancer immunotherapy either in the form of immune checkpoint blockades (ICBs), also known as immune checkpoint inhibitors (ICIs), or chimeric antigen receptor (CAR) T-cell therapy (3) has improved cancer treatment over traditional therapies including chemotherapy, surgery, radiation therapy, and so on. It shows stable clinical responses, progression free survival, and long survival benefits with a greater aim to reactivate the immune system to kill cancer cells (4).

Unfortunately, many cancer patients show resistance to immunotherapies either as primary or acquired resistance. Some patients are unresponsive to cancer immunotherapy treatment at early stages, known as primary resistance, while some patients manifest therapeutic resistance after 8 to 10 months, known as acquired resistance (5, 6). Different types of factors including immunosuppressive TME, high mitochondrial bioenergetics or oxidative metabolism, hypoxia, and tumor vasculature are mechanistically involved in inducing and promoting resistance to immunotherapy (7–9).

Various components of TME such as tumor cells, blood cells, and stromal cells stimulate cancer cells proliferation, migration, and metastasis, affect the infiltration of cytotoxic T cells into cancer cells, and consequently help cancer cells evade the immune surveillance mechanism (3, 10). These cells promote CD8+ T cell exhaustion, immunosuppressive environment, and immunoresistance by releasing proinflammatory cytokines, triggering angiogenesis and hypoxia and up-regulating immune checkpoint proteins (ICPs) such as programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin mucin -3 (TIM-3) (11–13). Thus, an interest has grown in the better understanding of the mechanism that modulates TME into immunosuppressive TME.

Cancer cells need high energy for their unregulated cell growth, progression, and metastasis, which cause them to use high mitochondrial bioenergetics and excessive oxygen compared to normal cells (14). Heme plays a central role in mitochondrial respiration by supplying the prosthetic groups of oxidative phosphorylation (OXPHOS) complexes (15). It had been found that cancer cells upregulate mitochondrial respiration, hence uptake more heme compared to normal cells. High ATP demand as well as enhanced oxygen consumption by tumor cells create hypoxia in TME areas. This hypoxic condition makes immunosuppressive TME by upregulating vascular endothelial growth factor (VEGF), resulting in neo-vasculature and activating hypoxia-inducible factor-1 (HIF-1) signaling pathways involved in therapeutic resistance (16, 17).

Hypoxia caused by rapidly proliferating tumor cells triggers the formation of new blood vessels, neo-vascularization, from existing blood vessels into TME, which is functionally and structurally abnormal compared to normal vessels (18). This disorganized tumor vasculature also inhibits the effective delivery of immunotherapeutic agents and other drugs to tumor areas and prevents the infiltration of cytotoxic CD8+ T cells, hence supporting cancer cells survival and progression with limited sensitivity to therapeutics (19).

Therefore, an investigation of the mechanism of an immunosuppressive TME amplified by heme and dysregulated mitochondrial respiration are required to improve the recruitment of effector immune cells into tumor areas, enhance anti-tumor immunity, and resensitize cancer cells to immunotherapy. This review will focus on the role of heme and mitochondrial respiration on mediating immunosuppressive TME and present the promising therapeutic prospects of targeting heme and mitochondrial respiration to normalize TME and potentiate immunotherapy.

Immunotherapy has emerged in the last decades as an exciting and promising new modality of cancer treatment and is considered as the “fifth pillar” of cancer therapy. It has a long history, dating back to William Bradley Coley’s use of Streptococcus pyogenes and Serratia marcescens, known as “Coley’s toxins”, which were injected into tumors to treat a variety of malignancies including sarcoma and lymphomas, and this cemented his place as the “father of immunotherapy” (20, 21). However, the modern use of immunotherapies in cancer can be traced back to Dr. James Allison and Dr. Tasuku Honjo’s work on immune checkpoint molecules, culminating in the first ICIs, ipilimumab and nivolumab, and earning the researchers the 2018 Nobel Prize in Medicine or Physiology (22). Immune checkpoint molecules are important proteins that modulate the immune response and allow cancer cells to evade anti-tumor immunity. ICIs are antibodies that target these ICPs to block interaction between ICPs and allow the body to resume an immune response capable of targeting the cancer. The US Food and Drug Administration (FDA) has approved three categories of ICIs such as anti-PD-1 (Nivolumab, Pembrolizumab, and Cemiplimab), anti-PD-L1 (Atezolimumab, Durvalumab and Avelum), and anti-CTLA-4 (Ipilimumab) (4). ICIs targeting CTLA-4, PD-1, and PD-L1 are used for different cancers, including melanomas, breast cancers, and lung cancers (23). The use of ICIs has helped thousands of patients to achieve a stable remission of their cancers.

Other forms of cancer immunotherapy include cytokines that can regulate the host immune response, such as interleukin -2 (IL-2) and interferons (IFNs). IL-2, which was first discovered in 1976, has been approved by FDA for kidney cancer and melanoma (24). IL-2 enhances the effects of T-cell and natural killer (NK) cell activity, allowing the host immune response to target cancers. It is also combined with other immunotherapies, such as ICIs, in order to boost the host immune response and allow targeting of “cold” tumors. Cold tumors are tumors that are poorly responsive to immunotherapy due to an immunosuppressive TME. IFNs work via a similar mechanism like interleukins and are also used in cancer immunotherapy (25). Another emerging immunotherapy treatment modality is CAR T-cell therapy enabling the use of the patients' own T-cells which are adapted to target specific cancers. T-cells are isolated from patients’ blood, and then T cells are edited to express a CAR that allows them to bind to specific tumor antigens. These modified CAR T-cells are then infused back into patient (26). There are currently 6 different CAR T-cell therapies approved by FDA to target a variety of blood cancers including acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (26). Cancer vaccines are another form of immunotherapy which can target a host immune response against antigens expressed by different cancers (27). They are generally divided into autologous, made from the patient’s own cells, and allogenic, made from artificial cells. These cancer vaccines hold promise for durable cancer response, including prevention of cancer (28). Immunotherapies have significantly improved the overall survival rate of patients, especially in case of melanoma cancers (29). In non-small cell lung cancer (NSCLC), pembrolizumab increased median progression-free survival to 10.3 months versus 6.0 months in chemotherapy group (30).

Unfortunately, many cancer patients show limited efficacy and adverse effects to immunotherapies due to different factors such as an immunosuppressive TME or mutations limiting the T cell infiltration to tumor cells (31). An adverse effects could be a cytokine storm, a sudden inflammatory response because of activating the immune system that can be dangerous to the host (32). Such adverse events have limited the possibilities of combining different immunotherapies as well as efficacy but significantly increased patients’ health risk. Additionally, not all cancers are responsive to immunotherapies, due to the presence of different immune evasion mechanisms and tumor heterogeneity phenotype in TME that allow TME to be immunosuppressive and resistance to immunotherapeutics. This is an area of major interest for researchers to target TME following combinatorial approaches such as combining immunotherapies with other agents and molecules to turn “cold” tumors into “hot” tumors favoring therapeutic responses to immunotherapy (32).

TME consists of cancer cells, tumor-antagonizing immune cells, such as effector T cells, dendritic cells (DC), NK cells, and tumor-promoting immune suppressive cells such as cancer associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), regulatory B cells (Bregs), and extracellular matrix (ECM) (33, 34). These cells and components of TME work together and their interaction mostly favor the growth of cancer, an immune evasion of cancer cells, and the insensitivity of cancer cells to immunotherapy (35). CAFs stimulate the release of cytokines and growth factors including platelet derived growth factor (PDGF), VEGF, hepatocyte growth factor (HGF), stromal cell-derived factor 1 (SDF1), and so on, which activates endothelial cells, resulting in an enhancement of tumor growth, angiogenesis process, and tumor vasculature. Immunosuppressive cells upregulate immunosuppressive cytokines such as IL-4, IL-10, IL-13 and transforming growth factor beta (TGF-β), stimulate angiogenesis by expressing VEGF and HIF-1α, and express ICPs, which are involved in tumor development, immunosuppressive TME formation, and anti-tumor immunity evasion (11–13, 36). Macrophage stimulates the release of tumor cells in the circulatory and lymphatic system and works as immunosuppressive agents through suppressing anti-tumor response and mechanism (37–39). TAMs are another prominent cancer promoting cells in TME. TAMs are present in two heterogenous form. One is pro-inflammatory M1 macrophage and another one is anti-inflammatory M2 macrophages, which are controlled and regulated by prostaglandin E2 (PE2), TGF-β, IL-4, IL-10, and IL-13, and involved in tumor-promoting activities by secreting anti-inflammatory cytokines such as TGF-β and IL-10 (40). To promote tumor growth and angiogenesis in TME, most of the macrophages in immune system are polarized to M2 macrophage, which suppress anti-tumor immune responses, subsequently releasing tumorigenic factors and promoting the remodulation of ECM, motility of tumor cells, and intravasation (41).

Cytotoxic T-lymphocytes (CTLs), also known as CD8+T cells, and helper T cells, also known as CD4+ T cells (TH1, TH2, and TH17), are the most prominent components of anti-cancer immunity (42, 43). Helper CD4+ T cells and cytotoxic CD8+ T cells mediate anti-tumor immunity through recognizing and killing cancer cells, but immunosuppressive activities within TME makes CTLs dysfunctional as well as causes T cells exhaustion (44, 45). Interaction between PD-1 and PD-L1, mainly expressed by T-cells and cancer cells, respectively, suppresses T-cells and stimulates the survival of cancer cells (46). Cancer immunotherapies mainly works by targeting T-cells and immune cells to restore anti-tumor immunity, promoting T-cells activation and T cell infiltration within the TME (47). Some constituents of TME have the potential to prevent tumor development, hence they are targeted for cancer immunotherapy. These cells in TME prevent tumor generation by releasing cytokine molecules as well as activating cytokines signaling pathways and direct cell contact (48). Many preclinical studies suggested that the inhibition of fibroblasts activating proteins in CAFs stimulate immunosuppression (49–51). Activation of CAFs by inhibiting different signaling pathways including nuclear factor kappa B (NF-kβ), C-X-C chemokine receptor 4 (CXCR4), fibroblast growth factor receptor (FGFR), and Hedgehog using selective inhibitors showed tumor suppressive or antitumor activity (52). CAFs also participate in an immunosuppressive function through suppressing cytotoxic T cell infiltration and function by releasing TGF-β (53). Targeting MDSCs in TME is another potential strategy for cancer immunotherapeutics. Preventing the immunosuppressive activities of MDSCs involves in blockage of MDSCs proliferation, recruitment, and differentiation of MDSCs into mature cells (54). Several studies showed that using inhibitors or neutralizing antibodies against tumor-derived factors like as IL-6, colony stimulating factor 1 (CSF1), and granulocyte-macrophage-colony stimulating factor (GM-CSF) and chemokines such as CXCR2, CXCR4, CC chemokine ligand 2 (CCL2) suppress MDSCs expansion and recruitment (55–59). Many synthetic inhibitors such as COX-2 inhibitors (celecoxib and acetylsalicylic acid), phosphodiesterase-5 inhibitors (tadalafil, sildenafil, and vardenafil) as well as bardoxolone are used to inhibit the immunosuppressive activities of MDSCs, eventually leading to the differentiation of MDSCs into non-suppressive mature cells (60–62).

However, tumor cells in TME remain in hypoxic state due to high oxidative metabolism and comparatively slow growth of blood vessels than the rapid growth of tumor cells. The simultaneous hypoxia and reoxygenation occurring in the TME areas causes the excessive production of reactive oxygen species (ROS) such as superoxide radical (O².-), hydroxyl radical (OH), singlet oxygen (O₂), and hydrogen peroxide (H₂O₂) which induce oxidative stress in the cells of TME that ultimately break both single and double DNA stand, initiate abnormal DNA synthesis, cause genetic instability, and finally generate aggressive tumor phenotype (63, 64). Although ROS plays a significant role in cancer progression, there are numerous studies suggesting that ROS also has negative effect on tumor growth. One study suggested that an excess level of ROS is produced during T cell activation by T cell receptor (TCR) signaling which regulates cell proliferation and clonal expansion and finally prevent progression of cancer and cancerous infection (65–68). ROS is also responsible for the development of cell-mediated and humoral immunity through the activation of B cell and T cell. H₂O₂ is a key element of ROS which has a significant role in B cell maturation and activation and enhance B cell receptor signaling (65, 69). ROS also controls tumor growth through creating balance between T cells and modulate T cell apoptosis (70). ROS from TME can spread up the phagocytosis process through recognizing and engulfing cancer cells (71). Moreover, phagocytic cells such as macrophage, monocytes, and neutrophils generate ROS, eventually leading to killing and clearance of damaged cells (72). ROS degrade pathogens through the formation of neutrophils extracellular traps by signaling cascade (73). In TME, excess ROS are also produced during the differentiation of macrophage (M1 and M2) (74, 75).

Hypoxia contributes to the generation of immunosuppressive TME and immunotherapeutic resistance by upregulating HIF-1α signaling pathways and triggering the expression of VEGF, PDGF, and other cytokines, resulting in a modification of tumor metabolism, angiogenesis, and lymphangiogenesis (76) (Figure 1). Tumor vasculature is structurally and functionally abnormal which leads to the uncontrolled proliferation of endothelial cells and the insufficient oxygen and nutrient supply to immune cells eventually blocking the function of T cells. Moreover, it interferes with the infiltration and delivery of functional T cells towards the tumor, downregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and develops the loss of antitumor immunity and immunotherapeutic resistance (77). Therefore, it is necessary to regulate TME to enhance the sensitivity of cancer cells to immunotherapy by understanding key mechanisms triggering hypoxia, tumor angiogenesis, and other processes mediating immunosuppression.

Various studies reported that cancer cells import elevated levels of heme and upregulate mitochondrial oxidative metabolism. These cause the modulation of TME into an immunosuppressive state that prevents antitumor immunity and supports cancer cells growth and proliferation, which are discussed into following sections and represented in Figure 1.

Cancer immunotherapy has been showing promising results and durable responses for lung cancer, breast cancer, head and neck cancer, and melanoma in various clinical trials studying the effects of blocking PD-1/PD-L1 interaction (118). However, resistance to immunotherapy is very common phenomenon to majority of cancer patients due to cancer heterogeneity and dynamic TME (35, 119). Different types of cells in TME interact with each other and create a hostile condition to anti-tumor immunity and cancer immunotherapy (35). In addition to making immunosuppressive TME, these mechanisms reduce the supply of oxygen and nutrients to immune cells, prevent the infiltration of effector T cells in tumor areas, and induce immunotherapeutic resistance (11). Therefore, it could be a promising approach to target heme and mitochondrial respiration for overcoming immunosuppressive mechanisms in TME and restoring anti-tumor immunity, which are discussed in the following section and summarized in Tables 1, 2.

Cancer immunotherapy targets the immune system to restore anti-tumor immunity and enable T cells to kill cancer cells; however, immunosuppressive TME limits the effects of cancer immunotherapy and immune responses (142). Hence, combinatorial approaches following the improvement of immunosuppressive TME and anti-tumor immunity could be useful to inhibit the progression of cancer, prevent immunotherapeutic resistance, and ensure sustainable therapeutic benefits with long-term prognosis. As immunotherapy agents mainly work on immune cells, combination of regimens that target different immunosuppressive mechanisms in TME might be useful to enhance the function of effector T cells, prevent the recruitment of immunosuppressive cells, and improve anti-tumor immunity. Many studies are currently ongoing that are aimed at targeting heme and mitochondrial respiration for overcoming therapeutic resistance and improving treatment outcomes. For instance, the use of IDO1 inhibitor is currently under therapeutic intervention as a cancer immunotherapy. This IDO1 inhibitor specifically targets IDO1, which is involved in cancer cells proliferation and immune suppression, and displaces heme from IDO1 by forming a complex (143, 144). It would be possible to improve the efficacy of cancer immunotherapy by regulating heme catabolism because of its role in inducing immunosuppressive characteristics. For example, Schillingmann et al. showed the potentiality of inhibiting HO-1 expression to improve adoptive T-cell responses against cancer patients producing wilms tumor protein -1 (WT1) (145). Synergistic effects of targeting heme catabolism and ICIs were observed in another study. Blockage of HO-1 expression and anti-PD-1 treatment together enhanced anti-tumor effects and anti-PD-1 treatment responses in melanoma mice models (121). However, the dependency of tumor cells and immune cells on oxidative metabolism is very complicated and immune cells need energy for their function, suggesting a judicious strategy for targeting mitochondrial OXPHOS (142). It had been found that the reprogramming of T cells metabolism by targeting mitochondrial OXPHOS increase tumor infiltrating cytotoxic T cells function, anti-tumor immunity, and cancer immunotherapy responses (146, 147). Combination of bezafibrate, which is an agonist of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha/peroxisome proliferator–activated receptors (PGC-1α/PPAR) complexes and selectively targets cytotoxic T cells oxidative metabolism, and anti-PD-L1 amplifies the number of T cells, effector T cells function, and anti-tumor immunity (148). It is also possible to epigenetically target T cells mitochondrial biogenesis, improve effector T cells function, and increase anti-tumor immunity found in an in vivo study (149). A number of OXPHOS inhibitors, such as metformin and canagliflozin, are actively being used in clinical trials examining their efficacy in combination with current cancer therapies (16). Notably, metformin combination therapy with anti-PD-1 immunotherapies, nivolumab and pembrolizumab, is being studied in several in-progress phase I and II clinical trials (16). Metformin acts by targeting mitochondrial complex I and thereby reducing OXPHOS, leading to reduced tumor hypoxia, which allows greater T-cell infiltration. Synergistic effects of metformin and IR-780 improved hypoxia by inhibiting mitochondrial respiration and delivering oxygen, respectively. This combination ultimately reduced immunosuppressive MDSC cells, cleared immunosuppressive TME, and enhanced anti-tumor responses (150). Scharping et al. showed that the treatment of metformin with anti-PD-1 reduces oxygen supply to tumor cells and increases the function of T cells (151). Hypoxia-activated prodrug TH-302 was studied in combination with anti-PD-1 or anti-CTLA-4 in prostate cancer mouse model. This combination was shown to reduce 80% tumor, hypoxia and the recruitment of immunosuppressive cells (152). Combinational treatments of IACS-010759, radiotherapy, and anti-PD-1 prevented resistance to anti-PD-1 and improved anti-tumor immunity in NSCLC adenocarcinoma xenograft mice models (153). Therefore, combinational treatments that target heme and mitochondrial OXPHOS along with the immune system could be a novel therapeutic approach by normalizing TME and improving anti-tumor immunity and responses to cancer immunotherapy. It would be desirable to study further in clinical settings to define mechanism and optimal doses, timing, and duration to which targeting heme, heme catabolism, and mitochondrial respiration could potentiate cancer immunotherapy. The heterogeneity of TME and individuals metabolic profiling, which affects therapeutic responses, should be considered before designing any drug regimens for better treatment outcomes and clinical responses. Moreover, some challenges are associated with developing metabolism based therapeutics, for instance, the dependency of both normal and cancer cells on metabolism, the rapid metabolic adaptation of cancer cells, drug delivery issues to mitochondria (154, 155), and so on, which should be addressed before developing anticancer therapies based on metabolic targets.

Immunosuppressive TME limits the success of cancer immunotherapy. Numerous studies have shown that heme, HO-1, and upregulated mitochondrial respiration play an important role in mediating immunosuppressive TME by promoting the recruitment of immunosuppressive cells, hypoxia, angiogenesis, and immune-evasion in TME areas. Various in vitro and in vivo studies show that targeting heme and mitochondrial respiration using heme sequestration molecules, HO-1 inhibitor, mitochondrial respiration inhibitor, and inhibitor of OXPHOS complexes could improve hypoxia, normalize vasculature, reduce oxygen consumption of cancer cells, and ultimately restore immunosupportive TME and anti-tumor immunity. However, much more attention is necessary to study the therapeutic effects of targeting heme and mitochondrial respiration and their combination with immunotherapeutic molecules, especially in clinical trials, which can help to develop effective immunotherapeutic drug combinations.

Conceptualization: ZA and LZ. Literature survey: ZA, N.S, and MA. Writing—original draft preparation: ZA, NS, and MA. Review and editing: ZA, NS, and LZ. Visualization: ZA, NS, and M.Y.A. Supervision: LZ. Funding acquisition: LZ. All authors contributed to the article and approved the submitted version.