Stimulating macropinocytosis of peptide-drug conjugates through DNA-dependent protein kinase inhibition for treating KRAS-mutant cancer.

KRAS-mutant cancers, due to their protein targeting complexity, present significant therapeutic hurdles. The identification of the macropinocytic phenotype in these cancers has emerged as a promising alternative therapeutic target. Our study introduces MPD1, an macropinocytosis-targeting peptide-drug conjugates (PDC), which is developed to treat KRAS mutant cancers. This PDC is specifically designed to trigger a positive feedback loop through its caspase-3 cleavable characteristic. However, we observe that this loop is hindered by DNA-PK mediated DNA damage repair processes in cancer cells. To counter this impediment, we employ AZD7648, a DNA-PK inhibitor. Interestingly, the combined treatment of MPD1 and AZD7648 resulted in a 100% complete response rate in KRAS-mutant xenograft model. We focus on the synergic mechanism of it. We discover that AZD7648 specifically enhances macropinocytosis in KRAS-mutant cancer cells. Further analysis uncovers a significant correlation between the increase in macropinocytosis and PI3K signaling, driven by AMPK pathways. Also, AZD7648 reinforces the positive feedback loop, leading to escalated apoptosis and enhanced payload accumulation within tumors. AZD7648 possesses broad applications in augmenting nano-sized drug delivery and preventing DNA repair resistance. The promising efficacy and evident synergy underscore the potential of combining MPD1 with AZD7648 as a strategy for treating KRAS-mutant cancers.

Organ-on-a-Chip Approach for Accelerating Blood-Brain Barrier Nanoshuttle Discovery.

Organ-on-a-chip, which recapitulates the dynamics of in vivo vasculature, has emerged as a promising platform for studying organ-specific vascular beds. However, its practical advantages in identifying vascular-targeted drug delivery systems (DDS) over traditional in vitro models remain underexplored. This study demonstrates the reliability and efficacy of the organ-on-a-chip in screening efficient DDS by comparing its performance with that of a conventional transwell, both designed to simulate the blood-brain barrier (BBB). The BBB nanoshuttles discovered through BBB Chip-based screening demonstrated superior functionality in vivo compared to those identified using transwell methods. This enhanced effectiveness is attributed to the BBB Chip's accurate replication of the structure and dynamics of the endothelial glycocalyx, a crucial protective layer within blood vessels, especially under shear stress. This capability of the BBB Chip has enabled the identification of molecular shuttles that efficiently exploit the endothelial glycocalyx, thereby enhancing transendothelial transport efficacy. Our findings suggest that organ-on-a-chip technology holds considerable promise for advancing research in vascular-targeted DDS due to its accurate simulation of molecular transport within endothelial systems.

Immune modulation of the liver metastatic colorectal cancer microenvironment via the oral CAPOX-mediated cGAS-STING pathway.

We evaluated modulation of the immunosuppressive tumor microenvironment in both local and liver metastatic colorectal cancer (LMCC), focusing on tumor-associated macrophages, which are the predominant immunosuppressive cells in LMCC. We developed an orally administered metronomic chemotherapy regimen, oral CAPOX. This regimen combines capecitabine and a nano-micelle encapsulated, lysine-linked deoxycholate and oxaliplatin complex (OPt/LDC-NM). The treatment effectively modulated immune cells within the tumor microenvironment by activating the cGAS-STING pathway and inducing immunogenic cell death. This therapy modulated immune cells more effectively than did capecitabine monotherapy, the current standard maintenance chemotherapy for colorectal cancer. The macrophage-modifying effect of oral CAPOX was mediated via the cGAS-STING pathway. This is a newly identified mode of immune cell activation induced by metronomic chemotherapy. Moreover, oral CAPOX synergized with anti-PD-1 antibody (αPD-1) to enhance the T-cell-mediated antitumor immune response. In the CT26. CL25 subcutaneous model, combination therapy achieved a 91 % complete response rate with a confirmed memory effect against the tumor. This combination also altered the immunosuppressive tumor microenvironment in LMCC, which αPD-1 monotherapy could not achieve. Oral CAPOX and αPD-1 combination therapy outperformed the maximum tolerated dose for treating LMCC, suggesting metronomic therapy as a promising strategy.

Neutrophil extracellular traps promote ΔNp63+ basal cell hyperplasia in chronic rhinosinusitis.

BACKGROUND: Neutrophil extracellular traps (NETs) are observed in chronic rhinosinusitis (CRS), although their role remains unclear. OBJECTIVES: This study aimed to investigate the influence of NETs on the CRS epithelium. METHODS: Forty-five sinonasal biopsy specimens were immunofluorescence-stained to identify NETs and p63+ basal stem cells. Investigators treated human nasal epithelial cells with NETs and studied them with immunofluorescence staining, Western blotting, and quantitative real-time PCR. NET inhibitors were administered to a murine neutrophilic nasal polyp model. RESULTS: NETs existed in tissues in patients with CRS with nasal polyps, especially in noneosinophilic nasal polyp tissues. p63+ basal cell expression had a positive correlation with the release of NETs. NETs induced the expansion of Ki-67+p63+ cells. We found that ΔNp63, an isoform of p63, was mainly expressed in the nasal epithelium and controlled by NETs. Treatment with deoxyribonuclease (DNase) I or Sivelestat (NET inhibitors) prevented the overexpression of ΔNp63+ epithelial stem cells and reduced polyp formation. CONCLUSIONS: These results reveal that NETs are implicated in CRS pathogenesis via basal cell hyperplasia. This study suggests a novel possibility of treating CRS by targeting NETs.

Biofunctional Inorganic Layered Double Hydroxide Nanohybrid Enhances Immunotherapeutic Effect on Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a widespread, recurrent, and chronic inflammatory skin condition that imposes a major burden on patients. Conventional treatments, such as corticosteroids, are associated with various side effects, underscoring the need for innovative therapeutic approaches. In this study, the possibility of using indole-3-acetic acid-loaded layered double hydroxides (IAA-LDHs) is evaluated as a novel treatment for AD. IAA is an auxin-class plant hormone with antioxidant and anti-inflammatory effects. Following the synthesis of IAA-LDH nanohybrids, their ability to induce M2-like macrophage polarization in macrophages obtained from mouse bone marrow is assessed. The antioxidant activity of IAA-LDH is quantified by assessing the decrease in intracellular reactive oxygen species levels. The anti-inflammatory and anti-atopic characteristics of IAA-LDH are evaluated in a mouse model of AD by examining the cutaneous tissues, immunological organs, and cells. The findings suggest that IAA-LDH has great therapeutic potential as a candidate for AD treatment based on its in vitro and in vivo modulation of AD immunology, enhancement of macrophage polarization, and antioxidant activity. This inorganic drug delivery technology represents a promising new avenue for the development of safe and effective AD treatments.

Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the critical components accelerating angiogenesis and tissue regeneration by promoting the migration of dermal fibroblasts and endothelial cells associated with matrix formation and remodeling in wound healing process. However, clinical applications of bFGF are substantially limited by its unstable nature due to rapid decomposition under physiological microenvironment. RESULTS: In this study, we present the bFGF-loaded human serum albumin nanoparticles (HSA-bFGF NPs) as a means of enhanced stability and sustained release platform during tissue regeneration. Spherical shape of the HSA-bFGF NPs with uniform size distribution (polydispersity index < 0.2) is obtained via a simple desolvation and crosslinking process. The HSA-bFGF NPs securely load and release the intact soluble bFGF proteins, thereby significantly enhancing the proliferation and migration activity of human dermal fibroblasts. Myofibroblast-related genes and proteins were also significantly down-regulated, indicating decrease in risk of scar formation. Furthermore, wound healing is accelerated while achieving a highly organized extracellular matrix and enhanced angiogenesis in vivo. CONCLUSION: Consequently, the HSA-bFGF NPs are suggested not only as a delivery vehicle but also as a protein stabilizer for effective wound healing and tissue regeneration.

Reprogramming the tumor microenvironment with biotechnology.

The tumor microenvironment (TME) is a unique environment that is developed by the tumor and controlled by tumor-induced interactions with host cells during tumor progression. The TME includes immune cells, which can be classified into two types: tumor- antagonizing and tumor-promoting immune cells. Increasing the tumor treatment responses is associated with the tumor immune microenvironment. Targeting the TME has become a popular topic in research, which includes polarizing macrophage phenotype 2 into macrophage phenotype 1 using Toll-like receptor agonists with cytokines, anti-CD47, and anti-SIPRα. Moreover, inhibiting regulatory T cells through blockades and depletion restricts immunosuppressive cells in the TME. Reprogramming T cell infiltration and T cell exhaustion improves tumor infiltrating lymphocytes, such as CD8+ or CD4+ T cells. Targeting metabolic pathways, including glucose, lipid, and amino acid metabolisms, can suppress tumor growth by restricting the absorption of nutrients and adenosine triphosphate energy into tumor cells. In conclusion, these TME reprogramming strategies exhibit more effective responses using combination treatments, biomaterials, and nanoparticles. This review highlights how biomaterials and immunotherapy can reprogram TME and improve the immune activity.

Protein-based nanocages for vaccine development.

Protein nanocages have attracted considerable attention in various fields of nanomedicine due to their intrinsic properties, including biocompatibility, biodegradability, high structural stability, and ease of modification of their surfaces and inner cavities. In vaccine development, these protein nanocages are suited for efficient targeting to and retention in the lymph nodes and can enhance immunogenicity through various mechanisms, including excellent uptake by antigen-presenting cells and crosslinking with multiple B cell receptors. This review highlights the superiority of protein nanocages as antigen delivery carriers based on their physiological and immunological properties such as biodistribution, immunogenicity, stability, and multifunctionality. With a focus on design, we discuss the utilization and efficacy of protein nanocages such as virus-like particles, caged proteins, and artificial caged proteins against cancer and infectious diseases such as coronavirus disease 2019 (COVID-19). In addition, we summarize available knowledge on the protein nanocages that are currently used in clinical trials and provide a general outlook on conventional distribution techniques and hurdles faced, particularly for therapeutic cancer vaccines.

Ferritin - a multifaceted protein scaffold for biotherapeutics.

The ferritin nanocage is an endogenous protein that exists in almost all mammals. Its hollow spherical structure that naturally stores iron ions has been diversely exploited by researchers in biotherapeutics. Ferritin has excellent biosafety profiles, and the nanosized particles exhibit rapid dispersion and controlled/sustained release pharmacokinetics. Moreover, the large surface-to-volume ratio and the disassembly/reassembly behavior of the 24 monomer subunits into a sphere allow diverse modifications by chemical and genetic methods on the surface and inner cage of ferritin. Here, we critically review ferritin and its applications. We (i) introduce the application of ferritin in drug delivery; (ii) present an overview of the use of ferritin in imaging and diagnosis for biomedical purposes; (iii) discuss ferritin-based vaccines; and (iv) review ferritin-based agents currently in clinical trials. Although there are no currently approved drugs based on ferritin, this multifunctional protein scaffold shows immense potential in drug development in diverse categories, and ferritin-based drugs have recently entered phase I clinical trials. This golden shortlist of recent developments will be of immediate benefit and interest to researchers studying ferritin and other protein-based biotherapeutics.

Feedback amplification of senolysis using caspase-3-cleavable peptide-doxorubicin conjugate and 2DG.

Therapy-induced senescence (TIS), a common outcome of current cancer therapy, is a known cause of late recurrence and metastasis and thus its eradication is crucial for therapy success. In this study, we introduced a conceptually novel strategy combining radiation-induced apoptosis-targeted chemotherapy (RIATC) with an effective glycolysis inhibitor, 2-deoxy-d-glucose (2DG) to target TIS. RIATC releases cytotoxic payload by amplification, continually increasing TIS, and this can be targeted by 2DG that stimulates an intrinsic apoptotic pathway in senescent cells, the senolysis; the senolytic 2DG also sensitizes cancer cells to chemo/radiation treatment. Anti-tumor efficacy of RIATC was investigated in numerous tumor models, and various cancer types were screened for TIS. Furthermore, in vitro evaluations of molecular markers of senescence, such as senescence-associated ß-galactosidase (SA-ß-Gal) assay, were performed to confirm that TIS was induced by RIATC therapy in MCF-7 cells. The combination therapy with 2DG proved to be effective in MCF-7 tumor-bearing mice that demonstrated feedback amplification of senolysis and successful inhibition of tumor growth. Our findings suggest that RIATC, when given together with 2DG, can overcome therapy-induced senescence and this combination is a promising strategy that enhances the therapeutic benefit of anti-cancer cytotoxic therapy.

A novel oral metronomic chemotherapy provokes tumor specific immunity resulting in colon cancer eradication in combination with anti-PD-1 therapy.

In this study, we investigated the immune-modulating effects of a novel metronomic chemotherapy (MCT) featuring combined oral oxaliplatin (OXA) and pemetrexed (PMX) for colon cancer. OXA and PMX were ionically complexed with lysine derivative of deoxycholic acid (DCK), and incorporated into nanoemulsions or colloidal dispersions, yielding OXA/DCK-NE and PMX/DCK-OP, respectively, to improve their oral bioavailabilities. MCT was not associated with significant lymphotoxicity whereas the maximum tolerated dose (MTD) afforded systemic immunosuppression. MCT was associated with more immunogenic cell death and tumor cell MHC-class I expression than was MTD. MCT improved the tumor antigen presentation of dendritic cells and increased the number of functional T cells in the tumor. MCT also helped to enhance antigen-specific memory responses both locally and systemically. By combining MCT with anti-programmed cell death protein-1 (αPD-1) therapy, the tumor volume was suppressed by 97.85 ± 84.88% compared to the control, resulting in a 95% complete response rate. Upon re-challenge, all tumor-free mice rejected secondary tumors, indicating the induction of a tumor specific memory response. Thus, MCT using an OXA and PMX combination, together with αPD-1, successfully treated colon cancer by activating both innate and adaptive immune cells and elicited tumor-specific long-term immune memory while avoiding toxicity caused by MTD treatment.

Immune cell targeting nanoparticles: a review.

Immune cells are attractive targets for therapy as they are direct participants in a variety of diseases. Delivering a therapeutic agent only to cells that act on a disease by distinguishing them from other cells has the advantage of concentrating the therapeutic effect and lowering systemic side effects. Distinguishing each immune cell from other immune cells to deliver substances, including drugs and genes, can be achieved using nanotechnology. And also nanoparticles can ensure in vivo stability and sustained drug release. In addition, there is an ease of surface modification, which is an important characteristic that can be utilized in targeted drug delivery systems. This characteristic allows us to utilize various properties that are specifically expressed in each immune cell. A number of studies have delivered various substances specifically to immune cells through surface engineering with active target ligands that can target each immune cell and enzyme-responsive coating, and demonstrated high therapeutic effects compared to conventional treatments. Progress in research on target delivery has been suggested to be a breakthrough for the treatments of various diseases, including cancer treatment.

Caspase-cleavable peptide-doxorubicin conjugate in combination with CD47-antagonizing nanocage therapeutics for immune-mediated elimination of colorectal cancer.

Here we report a novel combination of a caspase-cleavable peptide-doxorubicin conjugate (MPD-1) with CD47-antagonizing nanocage therapeutics for the treatment of microsatellite-stable (MSS) colorectal cancer (CRC). MPD-1 (i) upregulated markers of immunogenic cell death (ICD) in tumor, and increased co-stimulatory markers on dendritic cells (DCs), (ii) enhanced CD8+ T cell infiltration and antigen presenting cell (APC) activation, and (iii) showed negligible off-target immune-related toxicity compared to free dox. Then, the CD47 antagonist FS nanocage, a SIRPα-expressing ferritin nanocage, was co-administered with MPD-1 that resulted in 95.2% (p < 0.001) tumor growth inhibition in an established CRC model. T cell-mediated elimination of tumors was also confirmed by the tumor-specific activation of T cells detected by IFNγ and tumor-free mice were observed (95%) that bared a memory response when re-challenged. The strategically developed MPD-1 is an ideal adjuvant to immunotherapy and the combination with FS nanocage triggers potent immunity against MSS CRC. In summary, we present an approach to initiate and stimulate immune-mediated eradication of cancer cells using synergistic immunogenic agents targeting the MSS CRC.

Anticoagulation therapy promotes the tumor immune-microenvironment and potentiates the efficacy of immunotherapy by alleviating hypoxia.

PURPOSE: Here, this study verifies that cancer-associated thrombosis (CAT) accelerates hypoxia, which is detrimental to the tumor immune microenvironment by limiting tumor perfusion. Therefore, we designed an oral anticoagulant therapy to improve the immunosuppressive tumor microenvironment and potentiate the efficacy of immunotherapy by alleviating tumor hypoxia. EXPERIMENTAL DESIGN: A novel oral anticoagulant (STP3725) was developed to consistently prevent CAT formation. Tumor perfusion and hypoxia were analyzed with or without treating STP3725 in wild-type and P selectin knockout mice. Immunosuppressive cytokines and cells were analyzed to evaluate the alteration of the tumor microenvironment. Effector lymphocyte infiltration in tumor tissue was assessed by congenic CD45.1 mouse lymphocyte transfer model with or without anticoagulant therapy. Finally, various tumor models including K-Ras mutant spontaneous cancer model were employed to validate the role of the anticoagulation therapy in enhancing the efficacy of immunotherapy. RESULTS: CAT was demonstrated to be one of the perfusion barriers, which fosters immunosuppressive microenvironment by accelerating tumor hypoxia. Consistent treatment of oral anticoagulation therapy was proved to promote tumor immunity by alleviating hypoxia. Furthermore, this resulted in decrease of both hypoxia-related immunosuppressive cytokines and myeloid-derived suppressor cells while improving the spatial distribution of effector lymphocytes and their activity. The anticancer efficacy of αPD-1 antibody was potentiated by co-treatment with STP3725, also confirmed in various tumor models including the K-Ras mutant mouse model, which is highly thrombotic. CONCLUSIONS: Collectively, these findings establish a rationale for a new and translational combination strategy of oral anticoagulation therapy with immunotherapy, especially for treating highly thrombotic cancers. The combination therapy of anticoagulants with immunotherapies can lead to substantial improvements of current approaches in the clinic.

Impact of the conjugation of antibodies to the surfaces of polymer nanoparticles on the immune cell targeting abilities.

Antibodies have been widely used to provide targeting ability and to enhance bioactivity owing to their high specificity, availability, and diversity. Recent advances in biotechnology and nanotechnology permit site-specific engineering of antibodies and their conjugation to the surfaces of nanoparticles (NPs) in various orientations through chemical conjugations and physical adhesions. This study proposes the conjugation of poly(lactic-co-glycolic acid) (PLGA) NPs with antibodies by using two distinct methods, followed by a comparison between the cell-targeting efficiencies of both techniques. Full-length antibodies were conjugated to the PLGA-poly(ethylene glycol)-carboxylic acid (PLGA-PEG-COOH) NPs through the conventional carbodiimide coupling reaction, and f(ab')2 antibody fragments were conjugated to the PLGA-poly(ethylene glycol)-maleimide(PLGA-PEG-Mal) NPs through interactions between the f(ab')2 fragment thiol groups and the maleimide located on the nanoparticle surface. The results demonstrate that the PLGA nanoparticles conjugated with the f(ab')2 antibody fragments had a higher targeting efficiency in vitro and in vivo than that of the PLGA nanoparticles conjugated with the full-length antibodies. The results of this study can be built upon to design a delivery technique for drugs through biocompatible nanoparticles.

Dual mechanistic TRAIL nanocarrier based on PEGylated heparin taurocholate and protamine which exerts both pro-apoptotic and anti-angiogenic effects.

The selective cytotoxicity of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to cancer cells but not to normal cells makes it an attractive candidate for cancer therapeutics. However, the disadvantages of TRAIL such as physicochemical instability and short half-life limit its further clinical applications. In this study, TRAIL was encapsulated into a novel anti-angiogenic nanocomplex for both improved drug distribution at the tumor site and enhanced anti-tumor efficacy. A nanocomplex was prepared firstly by entrapping TRAIL into PEG-low molecular weight heparin-taurocholate conjugate (LHT7), which is previously known as a potent angiogenesis inhibitor. Then, protamine was added to make a stable form of nanocomplex (PEG-LHT7/TRAIL/Protamine) by exerting electrostatic interactions. We found that entrapping TRAIL into the nanocomplex significantly improved both pharmacokinetic properties and tumor accumulation rate without affecting the tumor selective cytotoxicity of TRAIL. Furthermore, the anti-tumor efficacy of nanocomplex was highly augmented (73.77±4.86%) compared to treating with only TRAIL (18.49 ± 19.75%), PEG-LHT7/Protamine (47.84 ± 14.20%) and co-injection of TRAIL and PEG-LHT7/Protamine (56.26 ± 9.98%). Histological analysis revealed that treatment with the nanocomplex showed both anti-angiogenic efficacy and homogenously induced cancer cell apoptosis, which suggests that accumulated TRAIL and LHT7 in tumor tissue exerted their anti-tumor effects synergistically. Based on this study, we suggest that PEG-LHT7/Protamine complex is an effective nanocarrier of TRAIL for enhancing drug distribution as well as improving anti-tumor efficacy by exploiting the synergistic mechanism of anti-angiogenesis.

Exosomes and Cancer Stem Cells in Cancer Immunity: Current Reports and Future Directions.

Cancer stem cells (CSCs), which have the capacity to self-renew and differentiate into various types of cells, are notorious for their roles in tumor initiation, metastasis, and therapy resistance. Thus, underlying mechanisms for their survival provide key insights into developing effective therapeutic strategies. A more recent focus has been on exosomes that play a role in transmitting information between CSCs and non-CSCs, resulting in activating CSCs for cancer progression and modulating their surrounding microenvironment. The field of CSC-derived exosomes (CSCEXs) for different types of cancer is still under exploration. A deeper understanding and further investigation into CSCEXs' roles in tumorigenicity and the identification of novel exosomal components are necessary for engineering exosomes for the treatment of cancer. Here, we review the features of CSCEXs, including surface markers, cargo, and biological or physiological functions. Further, reports on the immunomodulatory effects of CSCEXs are summarized, and exosome engineering for CSC-targeting is also discussed.

Brimonidine-montmorillonite hybrid formulation for topical drug delivery to the eye.

Brimonidine (BMD) is often prescribed as an eye drop to reduce the intraocular pressure (IOP) for glaucoma treatment. However, eye drops are limited by rapid clearance from the preocular surface, and hence a low ocular drug bioavailability. Therefore, in this study, we propose montmorillonite (MMT), as a delivery carrier, hybridized with BMD (BMD-MMT) for topical drug delivery to the eye. The BMD-MMT hybrid was prepared by intercalating the BMD molecules in the interlayer space of the MMT lattice via ion-exchange reaction; it was then formulated with polyvinyl alcohol (PVA) to produce a dry tablet (i.e., BMD-MMT@PVA). The BMD-MMT@PVA hybrid drug released BMD in a sustained manner for more than 5 h under in vitro conditions. When the hybrid drug was administered to rabbit eyes in vivo, 43% and 18.5% BMD-MMT still remained on the preocular surface for 10 and 60 min after administration, respectively. Thus, the BMD-MMT@PVA hybrid drug exhibited a prolonged decrease in IOP, that is, for 12 h, which was approximately two times longer than that observed with the commercially available BMD eye drop, Alphagan® P.

Modulating tumor immunity by metronomic dosing of oxaliplatin incorporated in multiple oral nanoemulsion.

In this study, a system for oral delivery of oxaliplatin (OXA) was prepared for metronomic chemotherapy to enhance antitumor efficacy and modulate tumor immunity. OXA was complexed with Nα-deoxycholyl-l-lysyl-methylester (DCK) (OXA/DCK) and formulated as a nanoemulsion (OXA/DCK-NE). OXA/DCK-NE showed 3.35-fold increased permeability across a Caco-2 cell monolayer, resulting in 1.73-fold higher oral bioavailability than free OXA. In addition, treatment of the B16F10.OVA cell line with OXA/DCK-NE resulted in successful upregulation of immunogenic cell death (ICD) markers both in vitro and in vivo. In a B16F10.OVA tumor-bearing mouse model, treatment with OXA/DCK-NE substantially impeded tumor growth by 63.9 ± 13.3% compared to the control group, which was also greater than the intravenous (IV) OXA group. Moreover, treatment with a combination of oral OXA/DCK-NE and anti-programmed cell death protein-1 (αPD-1) antibody resulted in 78.3 ± 9.67% greater inhibition compared to controls. More important, OXA/DCK-NE alone had immunomodulatory effects, such as enhancement of tumor antigen uptake, activation of dendritic cells in tumor-draining lymph nodes, and augmentation of both the population and function of immune effector cells in tumor tissue as well as in the spleen; no such effects were seen in the OXA IV group. These observations provide a rationale for combining oral metronomic OXA with immunotherapy to elicit synergistic antitumor effects.