Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses.

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.

Silencing adenosine A2a receptor enhances dendritic cell-based cancer immunotherapy.

Overexpression of adenosine in the tumor region leads to suppression of various immune cells, particularly T cells through ligation with adenosine 2a receptor (A2aR). In this study, we intended to increase the efficacy of tumor lysate-loaded DC vaccine by silencing the expression of A2aR on T cells through the application of A2aR-specific siRNA-loaded PEG-chitosan-lactate (PCL) nanoparticles (NPs) in the 4T1 breast tumor-bearing mice. Combination therapy by DC vaccine and siRNA-loaded NPs markedly induced tumor regression and increased survival time of mice. These ameliorative effects were partly via downregulation of immunosuppressive cells, increased function of cytotoxic T lymphocytes, and induction of immune-stimulatory cytokines. Moreover, combination therapy could markedly suppress angiogenesis and metastasis processes. These results imply the efficacy of novel combination therapy for the treatment of breast cancer by using A2aR siRNA-loaded NPs and DC vaccine which can be translated into the initial phase of clinical trials in the near future.

Cancer associated fibroblasts as novel promising therapeutic targets in breast cancer.

Breast cancer is one of the most important women-related malignancies, which is incurable (particularly in advanced stages) and tumor microenvironment is a number one accused part in the inefficiency of current anti-breast cancer therapeutic strategies. The tumor microenvironment is composed of various cellular and acellular components, which provide an optimum condition for freely expanding cancer cells in various cancer types, particularly breast cancer. Cancer-associated fibroblasts (CAFs) are one of the main cell types in the breast tumor region, which can promote various tumor-promoting processes such as expansion, angiogenesis, metastasis and drug resistance. CAFs directly (by cell-to-cell communication) and indirectly (through secreting soluble factors) can exert their tumorigenic functions. We try to elucidate the immunobiology of CAFs, their origin, function, and heterogeneity in association with their role in various cancer-promoting processes in breast cancer. Based on current knowledge, we believe that the origin of CAFs, their subsets, and their specific expressed biomarkers determine their pro- or anti-tumor functions. Therefore, targeting CAF without considering their specific functions may lead to a deleterious outcome. We propose to find and characterize each subtype of CAFs in association with its specific function in different stages of breast cancer to develop novel promising therapeutic approaches against the right CAF subtype.

PD-L1/PD-1 axis as a potent therapeutic target in breast cancer.

Although both the incidence and the mortality rate of breast cancer is rising, there is no potent and practical option for the treatment of these patients, particularly in advanced stages. One of the most critical challenges for treatment is the presence of complicated and extensive tumor escape mechanisms in the tumor microenvironment. Immune checkpoint molecules are of the main immunosuppressive mechanisms used by cancerous cells to block anti-cancer immune responses. Among these molecules, PD-1 (Programmed cell death) and PD-L1 (programmed cell death-ligand 1) have been considered as worthy therapeutic targets for breast cancer therapy. In this review, we intend to discuss the immunobiology and signaling of the PD-1/PD-L1 axis and highlight its importance as a worthy therapeutic target in breast cancer. We believe that the prognostic value of PD-L1 depends on the breast cancer subtype. Moreover, the combination of PD-1/PD-L1 targeting with immune-stimulating vaccines can be considered as an effective therapeutic strategy in breast cancer.

Silencing of IL-6 and STAT3 by siRNA loaded hyaluronate-N,N,N-trimethyl chitosan nanoparticles potently reduces cancer cell progression.

The immunosuppressive nature of the tumor microenvironment is a critical problem that should be considered before the design of immunotherapies. Interleukin (IL)-6 and its related downstream molecules such as signal transducer and activator of transcription (STAT)3 play an important role in the cancer progression, which can be considered as potential therapeutic targets. In the present study, we generated the active-targeted hyaluronate (HA) recoated N, N, N-trimethyl chitosan (TMC) nanoparticles (NPs) to deliver IL-6- and STAT3-specific small interfering RNAs (siRNAs) to the CD44-expressing cancer cells. We utilized the interaction between HA and CD44 to increase the specificity and efficacy of cellular uptake in NPs. The results showed that the synthesized NPs had efficient physicochemical characteristics, high transfection efficiency, low toxicity, and controlled siRNA release. siRNA-loaded NPs significantly inhibited the IL-6/STAT3 expression, which was associated with blockade of proliferation, colony formation, migration, and angiogenesis in cancer cells. These findings imply the potential of HA-TMC NPs as potent vectors in gene therapy and their application for the silencing of IL-6 and STAT3, as a novel anti-cancer combination therapeutic strategy, for the first time.

Hypoxia inducible factors in the tumor microenvironment as therapeutic targets of cancer stem cells.

Cancer stem cells (CSC) constitute a small area of the tumor mass and are characterized by self-renewal, differentiation and the ability to promote the development of secondary chemo-resistant tumors. Self-renewal of CSCs is regulated through various signaling pathways including Hedgehog, Notch, and Wnt/ß-catenin pathways. A few surface markers have been identified, which provide a means of targeting CSCs according to tumor type. Depending on the proximity of CSCs to the tumor hypoxic niche, hypoxia-inducible factors (HIFs) can play a critical role in modulating several CSC-related characteristics. For instance, the upregulation of HIF-1 and HIF-2 at tumor sites, which correlates with the expansion of CSCs and poor cancer prognosis, has been demonstrated. In this review, we will discuss the mechanisms by which hypoxia enhances the development of CSCs in the tumor microenvironment. Targeting HIFs in combination with other common therapeutics is pre-requisite for effective eradication of CSCs.