FOXP3 (in)stability and cancer immunotherapy.

Dysregulation of regulatory T cells (Tregs) is described in the context of inflammatory and autoimmune diseases, and cancer. Forkhead box P3 (FOXP3) is a transcription factor that its activity is an indicator of Treg identity. FOXP3 induces metabolic versatility in intra-tumoral Tregs, so that its deficiency mediates Treg instability or even gives rise to the acquisition of effector T cell phenotype. FOXP3 dysregulation and defectiveness occurs upon ubiquitination, methylation and presumably acetylation. Stimulators of PTEN, mammalian target of rapamycin complex 2 (mTORC2), and nucleus accumbens-associated protein-1 (NAC1), and inhibitors of B lymphocyte-induced maturation protein-1 (Blimp-1), Deltex1 (DTX1) and ubiquitin-specific peptidase 22 (USP22) are suggested to hamper FOXP3 stability, and to promote its downregulation and further Treg depletion. A point is that Treg subsets reveal different reliance on FOXP3, which indicates that not all Tregs are strictly dependent on FOXP3, and presumably Tregs with different origin rely on diverse regulators of FOXP3 stability. The focus of this review is over the current understanding toward FOXP3, its activity in Tregs and influence from different regulators within tumor microenvironment (TME). Implication of FOXP3 targeting in cancer immunotherapy is another focus of this paper.

Immunotherapy of Human Melanoma: Past, Present, Future.

Immunotherapy with immune checkpoint inhibitors (ICIs) is a promising therapeutic schedule in advanced solid cancers. In this review, clinical trials from highly reputable journals are interpreted for safety and efficacy evaluation of the common anti-programmed death-1 (PD-1) inhibitor nivolumab and/or the most known anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibitor ipilimumab in advanced melanoma. Current progress in the field of melanoma immunotherapy is the focus of this review. Solo nivolumab and combo nivolumab-ipilimumab show higher responses compared to solo ipilimumab or chemotherapy. BRAF and programmed death-ligand 1 (PDL1) expression states are seemingly not reliable biomarkers of response to ICI therapy in melanoma. Solo ipilimumab and particularly a combination of nivolumab-ipilimumab show higher adverse events (AEs) compared with solo nivolumab or chemotherapy. Besides, ICI therapy is safer in mucosal melanoma, but its efficacy is higher in the cutaneous subtype. Patients receiving combination regimens who are experiencing serious AEs can discontinue such regimens until recovery and still maintain clinical benefits. To conclude, combo nivolumab-ipilimumab represents more therapeutic advantages compared with solo nivolumab or ipilimumab, but the rate of AEs is higher for combination regimens. Resistance to combo nivolumab-ipilimumab demands the application of novel approaches to go with ICIs in melanoma immunotherapy. Immunogenic agents, alternative immune checkpoints, vaccination, oncolytic viruses, extracellular vesicles (EVs) and fecal microbiome transplantation (FMT) are novel strategies in patients developing ICI resistance.

Selective targeting or reprogramming of intra-tumoral Tregs.

Regulatory T cells (Tregs) are critical immunosuppressive cells that are frequently present in the tumor microenvironment of solid cancers and enable progression of tumors toward metastasis. The cells expand in response to tumor-associated antigens and are actively involved in bypassing immunotherapy with immune checkpoint inhibitors through integrating numerous environmental signals. A point here is that Tregs are clonally distinct in peripheral blood from tumor area. Currently, an effective and novel task in cancer immunotherapy is to selectively destabilize or deplete intra-tumoral Tregs in order to avoid systemic inflammatory events. Helios is a transcription factor expressed selectively by Tregs and promotes their stabilization, and Trps1 is a master regulator of intra-tumoral Tregs. Anti-CCR8 and the IL-2Rßγ agonist Bempegaldesleukin selectively target intra-tumoral Treg population, with the former approved to not elicit autoimmunity. Disarming Treg-related immunosuppression in tumors through diverting their reprogramming or promoting naïve T cell differentiation into cells with effector immune activating profile is another promising area of research in cancer immunotherapy. Blimp-1 inhibitors and glucocorticoid-induced TNFR-related protein agonists are example approaches that can be used for diverting Treg differentiation into Th1-like CD4+ T cells, thereby powering immunogenicity against cancer. Finally, selective target of intra-tumoral Tregs and their reprogramming into effector T cells is applicable using low-dose chemotherapy, and high-salt and high-tryptophan diet.

An Update of Extracellular Vesicle Involvement in different Steps of Cancer Metastasis and Targeting Strategies.

Cancer metastasis is the deadliest event in tumorigenesis. Despite extensive research, there are still unsolved challenges regarding early metastasis detection and targeting strategies. Extracellular vesicles (EVs) and their impact on tumorigenic-related events are in the eye of current investigations. EVs represent a plethora of biomarkers and information, and they are considered key determinants in tumor progression and for tumor prognosis and monitoring. EVs are one of the key mediators for inter-cellular communications between tumor cells and their nearby stroma. They are involved in different steps of metastasis from invasion toward formation of pre-metastatic niches (PMNs), and final growth and colonization of tumor cells in desired organ/s of the target. Membrane components of EVs and their cargo can be traced for the identification of tumor metastasis, and their targeting is a promising strategy in cancer therapy. In this review, we aimed to discuss the current understanding of EV-based metastatic predilection in cancer, providing updated information about EV involvement in different metastatic steps and suggesting some strategies to hamper this devastating condition.

WNT/ß-catenin regulatory roles on PD-(L)1 and immunotherapy responses.

Dysregulation of WNT/ß-catenin is a hallmark of many cancer types and a key mediator of metastasis in solid tumors. Overactive ß-catenin signaling hampers dendritic cell (DC) recruitment, promotes CD8+ T cell exclusion and increases the population of regulatory T cells (Tregs). The activity of WNT/ß-catenin also induces the expression of programmed death-ligand 1 (PD-L1) on tumor cells and promotes programmed death-1 (PD-1) upregulation. Increased activity of WNT/ß-catenin signaling after anti-PD-1 therapy is indicative of a possible implication of this signaling in bypassing immune checkpoint inhibitor (ICI) therapy. This review is aimed at giving a comprehensive overview of the WNT/ß-catenin regulatory roles on PD-1/PD-L1 axis in tumor immune ecosystem, discussing about key mechanistic events contributed to the WNT/ß-catenin-mediated bypass of ICI therapy, and representing inhibitors of this signaling as promising combinatory regimen to go with anti-PD-(L)1 in cancer immunotherapy. Ideas presented in this review imply the synergistic efficacy of such combination therapy in rendering durable anti-tumor immunity.

Tumor-educated platelets.

Beyond traditional roles in homeostasis and coagulation, growing evidence suggests that platelets also reflect malignant transformation in cancer. Platelets are present in the tumor microenvironment where they interact with cancer cells. This interaction results in direct and indirect "education" as evident by platelet alterations in adhesion molecules, glycoproteins, nucleic acids, proteins and various receptors. Subsequently, these tumor-educated platelets (TEPs) circulate throughout the body and play pivotal roles in promotion of tumor growth and dissemination. Accordingly, platelet status can be considered a unique blood-based biomarker that can potentially predict prognosis and therapeutic success. Recently, liquid biopsies including TEPs have received much attention as safe, minimally invasive and sensitive alternatives for patient management. Herein, we provide an overview of TEPs and explore their benefits and limitations in cancer.

Liquid biopsy in colorectal cancer.

Liquid biopsy refers to a set of pathological samples retrieved from non-solid sources, such as blood, cerebrospinal fluid, urine, and saliva through non-invasive or minimally invasive approaches. In the recent decades, an increasing number of studies have focused on clinical applications and improving technological investigation of liquid biopsy biosources for diagnostic goals particularly in cancer. Materials extracted from these sources and used for medical evaluations include cells like circulating tumor cells (CTCs), tumor-educated platelets (TEPs), cell-free nucleic acids released by cells, such as circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cell-free RNA (cfRNA), and exosomes. Playing significant roles in the pathogenesis of human malignancies, analysis of these sources can provide easier access to genetic and transcriptomic information of the cancer tissue even better than the conventional tissue biopsy. Notably, they can represent the inter- and intra-tumoral heterogeneity and accordingly, liquid biopsies demonstrate strengths for improving diagnosis in early detection and screening, monitoring and follow-up after therapies, and personalization of therapeutical strategies in various types of human malignancies. In this review, we aim to discuss the roles, functions, and analysis approaches of liquid biopsy sources and their clinical implications in human malignancies with a focus on colorectal cancer.

Modifying CAR-T cells with anti-checkpoints in cancer immunotherapy: A focus on anti PD-1/PD-L1 antibodies.

Chimeric antigen receptor-modified T (CAR-T) are genetically engineered cells to express tumor-specific antigens revolutionizing the treatment of hematologic malignancies. The hostile tumor microenvironment (TME) remains a challenge for CAR-T cell therapy in solid tumors. As a solution, combinational therapy with immune checkpoint inhibitors (ICIs) is shown to improve the safety and efficacy of CAR-T cell therapy. To avoid side effects related to the application of ICIs in combinational therapy, engineering CARs to express tumor-specific antigens may help improvement of clinical outcomes. Those CARs expressing single chain variable fragments (scFvs) or nanobodies against immune checkpoint stimulatory or inhibitory molecules, such as the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis are being extensively studied in various clinical trials. In this review, we discuss the significance of anti-PD-(L)1 scFv-expressing CAR-T cells in the treatment of human cancers, describing current challenges and potential strategies to overcome such predicaments in the area of cancer immunotherapy.

Alternative immune checkpoints in immunoregulatory profile of cancer stem cells.

Tumor-mediated bypass of immune checkpoint inhibitor (ICI) therapy with anti-programmed death-1 (PD-1), anti-programmed death-ligand 1 (PD-L1, also called B7-H1 or CD274) or anti-cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is a challenge of current years in the area of cancer immunotherapy. Alternative immune checkpoints (AICs) are molecules beyond the common PD-1, PD-L1 or CTLA-4, and are upregulated in patients who show low/no ICI responses. These are members of B7 family including B7-H2 (ICOS-L), B7-H3 (CD276), B7-H4 (B7x), V-domain immunoglobulin suppressor of T cell activation (VISTA), B7-H6, HHLA2 (B7-H5/B7-H7) and catabolic enzymes like indoleamine 2,3-dioxygenase 1 (IDO1), and others that are also contributed to the regulation of tumor immune microenvironment (TIME). There is also strong evidence supporting the implication of AICs in regulation of cancer stemness and expanding the population of cancer stem cells (CSCs). CSCs display immunoregulatory capacity and represent multiple immune checkpoints either on their surface or inside. Besides, they are active promoters of resistance to the common ICIs. The aim of this review is to investigate interrelations between AICs with stemness and differentiation profile of cancer. The key message of this paper is that targeted checkpoints can be selected based on their impact on CSCs along with their effect on immune cells. Studies published so far mainly focused on immune cells as a target for anti-checkpoints. Ex vivo engineering of extracellular vesicles (EVs) equipped with CSC-targeted anti-checkpoint antibodies is without a doubt a key therapeutic target that can be under consideration in future research.

Dual anti-PD-(L)1/TGF-ß inhibitors in cancer immunotherapy - Updated.

Immune checkpoint inhibitor (ICI) therapy suffers from tumor resistance and relapse in majority of patients due to the suppressive tumor immune microenvironment (TIME). Advances in the field have brought about development of fusion proteins able to target two signaling simultaneously and to exert maximal anti-cancer immunity. Bispecific inhibitors of transforming growth factor (TGF)-ß signaling and programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1) are developed to reduce the rate of relapse and to achieve durable anti-cancer therapy. TGF-ß is well-known for its immunosuppressive activity, and it takes critical roles in promotion of all tumor hallmarks. Bispecific anti-PD-(L)1/TGF-ß inhibitors reinvigorate effector activity of CD8+ T and natural killer (NK) cells, hamper regulatory T cell (Treg) expansion, and increase the density of anti-tumor type 1 macrophages (M1). Responses to the bispecific approach are higher compared with solo anti-PD-(L)1 or TGF-ß targeted therapy, and are seemingly more pronounced in human papillomavirus (HPV)+ patients. High expression of PD-L1 or immune-excluded phenotype in a tumor can also be markers of better response to the bispecific strategy. Besides, anti-PD-(L)1/TGF-ß inhibitor therapy can be used safely with other therapeutic modalities including vaccination, radiation and chemotherapy.

Extracellular vesicle-based drug delivery in cancer immunotherapy.

Extracellular vesicles (EVs) are a group of nanoscale membrane-bound organelles including exosomes, microvesicles (MVs), membrane particles, and apoptotic bodies, which are released from almost all eukaryotic cells. Owing to their ingredients, EVs can be employed as biomarkers for human diseases. Interestingly, EVs show favorable features as candidates for targeted drug delivery and thus, they are suggested as ideal drug carriers as well as good vaccines for various human diseases including cancer. Among various drugs loaded in EVs for targeted drug delivery, immune checkpoint inhibitors (ICIs), including antibodies against programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), have attracted an increasing attention for cancer researchers and clinicians. Animal and clinical studies have shown combination of EVs and immunotherapy antibodies to improve the efficacy and reduce possible side effects in systemic administration of ICIs. In this review, we discuss the EVs and their significance in drug delivery with a focus on cancer immunotherapy agents.

The impact of PD-L1 as a biomarker of cancer responses to combo anti-PD-1/CTLA-4.

Combination therapy of solid tumors with immune checkpoint inhibitors (ICIs) is a promising and rapidly evolving area of clinical research. Combo nivolumab-ipilimumab therapy has demonstrated potent efficacy in recent years, and PD-L1 expression profile has shown to play a key role in determining the most optimal immunotherapeutic regimen in advanced cancer patients. Here, the focus is over the impact of PD-L1 on combo nivolumab-ipilimumab in advanced solid cancer patients. Interpretations of this review indicate that patient responses to combo nivolumab-ipilimumab can be affected from different levels of PD-L1 expression states. A point required attention is the variations in responses among diverse cancer types or between different doses of the immunotherapy drugs. In general, higher rates of responses are seen with higher PD-L1 expression levels in many cancer types. This, however, is not coincided with survival of patients. Taken all into consideration, it could be asserted that considering PD-L1 as a solo biomarker may not be reliable for predicting clinical efficacy of combo nivolumab-ipilimumab. Thus, a search for other biomarkers or combination of PD-L1 with other factors may be considered for predicting patient responses.

B7-H3 immunoregulatory roles in cancer.

B7 homolog 3 (B7-H3, also called CD276) is a checkpoint of B7 family that is aberrantly and consistently expressed in several human cancers, and its overexpression correlates with weak prognosis. B7-H3 is expressed on a number of cells, and it acts as a driver of immune evasion. This is mediated through hampering T cell infiltration and promoting exhaustion of CD8+ T cells. Increased B7-H3 activity also promotes macrophage polarity toward pro-tumor type 2 (M2) phenotype. In addition, high B7-H3 activity induces aberrant angiogenesis to promote hypoxia, a result of which is resistance to common immune checkpoint inhibitor (ICI) therapy. This is mediated through the impact of hypoxia on dampening CD8+ T cell recruitment into tumor area. The immunosuppressive property of B7-H3 offers insights into targeting this checkpoint as a desired approach in cancer immunotherapy. B7-H3 can be a target in blocking monoclonal antibodies (mAbs), combination therapies, chimeric antigen receptor-modified T (CAR-T) cells and bispecific antibodies.

Mechanisms of CD8+ T cell exclusion and dysfunction in cancer resistance to anti-PD-(L)1.

CD8+ T cells are the front-line defensive cells against cancer. Reduced infiltration and effector function of CD8+ T cells occurs in cancer and is contributed to defective immunity and immunotherapy resistance. Exclusion and exhaustion of CD8+ T cells are the two key factors associated with reduced durability of immune checkpoint inhibitor (ICI) therapy. Initially activated T cells upon exposure to chronic antigen stimulation or immunosuppressive tumor microenvironment (TME) acquire a hyporesponsive state that progressively lose their effector function. Thus, a key strategy in cancer immunotherapy is to look for factors contributed to defective CD8+ T cell infiltration and function. Targeting such factors can define a promising supplementary approach in patients receiving anti-programmed death-1 receptor (PD-1)/anti-programmed death-ligand 1 (PD-L1) therapy. Recently, bispecific antibodies are developed against PD-(L)1 and a dominant factor within TME, representing higher safety profile and exerting more desired outcomes. The focus of this review is to discuss about promoters of deficient infiltration and effector function of CD8+ T cells and their addressing in cancer ICI therapy.

Advances in dendritic cell vaccination therapy of cancer.

Traditionally, vaccines have helped eradication of several infectious diseases and also saved millions of lives in the human history. Those prophylactic vaccines have acted through inducing immune responses against a live attenuated, killed organism or antigenic subunits to protect the recipient against a real infection caused by the pathogenic microorganism. Nevertheless, development of anticancer vaccines as valuable targets in human health has faced challenges and requires further optimizations. Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) that play essential roles in tumor immunotherapies through induction of CD8+ T cell immunity. Accordingly, various strategies have been tested to employ DCs as therapeutic vaccines for exploiting their activity against tumor cells. Application of whole tumor cells or purified/recombinant antigen peptides are the most common approaches for pulsing DCs, which then are injected back into the patients. Although some hopeful results are reported for a number of DC vaccines tested in animal and clinical trials of cancer patients, such approaches are still inefficient and require optimization. Failure of DC vaccination is postulated due to immunosuppressive tumor microenvironment (TME), overexpression of checkpoint proteins, suboptimal avidity of tumor-associated antigen (TAA)-specific T lymphocytes, and lack of appropriate adjuvants. In this review, we have an overview of the current experiments and trials evaluated the anticancer efficacy of DC vaccination as well as focusing on strategies to improve their potential including combination therapy with immune checkpoint inhibitors (ICIs).

The impact of hypoxia on tumor-mediated bypassing anti-PD-(L)1 therapy.

Extending the durability of response is the current focus in cancer immunotherapy with immune checkpoint inhibitors (ICIs). However, factors like non-immunogenic tumor microenvironment (TME) along with aberrant angiogenesis and dysregulated metabolic systems are negative contributors. Hypoxia is a key TME condition and a critical promoter of tumor hallmarks. It acts on immune and non-immune cells within TME in order for promoting immune evasion and therapy resistance. Extreme hypoxia is a major promoter of resistance to the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitor therapy. Hypoxia inducible factor-1 (HIF-1) acts as a key mediator of hypoxia and a critical promoter of resistance to the anti-PD-(L)1. Targeting hypoxia or HIF-1 can thus be an effective strategy for reinvigoration of cellular immunity against cancer. Among various strategies presented so far, the key focus is over vascular normalization, which is an approach highly effective for reducing the rate of hypoxia, increasing drug delivery into the tumor area, and boosting the efficacy of anti-PD-(L)1.

HHLA2 immune-regulatory roles in cancer.

Human endogenous retrovirus H long terminal repeat-associating protein 2 (HHLA2 or B7-H7) is a newly discovered B7 family member. HHLA2 is aberrantly expressed in solid tumors and exerts co-stimulatory or co-inhibitory activities dependent on interaction with counter receptors. HHLA2 represents co-stimulatory effects upon interaction with transmembrane and immunoglobulin domain containing 2 (TMIGD2, also called CD28H), but its interaction with killer cell Ig-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3) renders co-inhibitory effects. TMIGD2 is mainly expressed on resting or naïve T cells, whereas expression of KIR3DL3 occurs on activated T cells. HHLA2/KIR3DL3 attenuates responses from both innate and adaptive anti-tumor immunity, and the activity within this axis is regarded as a biomarker of weak prognosis in cancer patients. HHLA2/KIR3DL3 promotes CD8+ T cell exhaustion and induces macrophage polarity toward pro-tumor M2 phenotype. HHLA2 represents diverse expression profile and activity in tumor and stroma. Tumoral expression of HHLA2 is presumably higher compared with programmed death-ligand 1 (PD-L1), and HHLA2 co-expression with PD-L1 is indicative of more severe outcomes. A suggested strategy in patients with HHLA2high cancer is to use monoclonal antibodies for specifically suppressing the HHLA2 inhibitory receptor KIR3DL3, not the HHLA2 ligand. TMIGD2 can be a target for development of agonistic bispecific antibodies for hampering tumor resistance to the programmed death-1 (PD-1)/PD-L1 blockade therapy.

B7x in cancer immunity and immunotherapy.

B7x (also called B7-H4) is a co-inhibitory molecule of B7 family that is highly expressed in non-inflamed or cold cancers, and its aberrant expression is contributed to cancer progression and poor outcomes. B7x preferentially expresses on antigen-presenting cells (APCs) and in tumor cells, and it acts as an alternative anti-inflammatory immune checkpoint for hampering peripheral immune responses. Augmented infiltration of immunosuppressive cells, reduced proliferation and effector function of CD4+ and CD8+ T cells, and increased generation of regulatory T cells (Tregs) are outcomes of increased B7x activity in cancer. Evaluation of B7x in sera can be exploited as an effective biomarker of response in cancer patients. B7x overexpression generally occurs in programmed death-ligand 1 (PD-L1)- cancers and is involved in tumor resistance to anti-programmed death-1 (PD-1), anti-PD-L1 or anti-cytotoxic T lymphocyte associated antigen-4 (CTLA-4) therapy. Co-expression of B7x receptor with PD-1 on CD8+ T cells has made the anti-B7x a fruitful approach for reinvigoration of the functionality of exhausted T cells and is served as a complementary regimen in patients who are irresponsive to the common immune checkpoint inhibitor (ICI) therapy. An advance in the field is the development of bispecific antibodies against B7x with other regulatory molecules within tumor microenvironment (TME).