Enhancing Radiotherapy for Hypoxic Tumors: Integrative Strategies Using Bacteria and Nanoparticles

Emerging nanozymes for potentiating radiotherapy and radiation protection

In a previous study we found both in vitro and in an orthotropic NSCLC model in SCID mice that survival of NSCLC cells in hypoxic microenvironment is dependent upon Notch-1 signaling. Active Notch-1 was expressed in hypoxic tumor microenvironment only. Notch-1 provides survival stimuli through exacerbated Akt-1 activation due to suppression of PTEN transcription and positive regulation of the transcription of IGF-1 and its receptor. Hypoxic tumor environment is a problem for NSCLC treatment because it is responsible for cancer resistance to chemotherapy, tumor recurrence and metastasization. Also, hypoxic tumor microenvironment is quiescent and represses mTORC-1 activity. We show a number of experiments aimed at targeting hypoxic tumor tissue in the orthotropic NSCLC model. We attempted to target the Notch-1/IGF-1R/Akt-1 axis using three agents (a γ-secretase inhibitor, MRK-003; a fully humanized antibody against the human IGF-1R that promotes the receptor degradation, MK-0646; a pan-Akt inhibitor, MK-2206, all drugs provided by Merck Inc.) alone or in various combinations. We also used in combination studies drugs that are used for the treatment of advanced NSCLC, namely cisplatin and Erlotinib. The orthotropic model was established using two cell lines non-dependent on Notch-3 signaling. siRNA to Notch-3 in A549 and H1437 affects neither the growth rate nor the survival of these cells. All treatments (besides MK-2206) significantly prolonged the median survival of mice compared to controls (16 mice/experimental arm). MRK-003 treatment yielded specific death of hypoxic tumor areas. The tumors excised from the mice reaching an end-point displayed a significant reduction of markers of hypoxia as assessed by RT-qPCR using human specific primers. Moreover, qPCR measurements of the ratio between human/mouse GAPDH in DNA extracted from the whole brains and livers showed a significant reduction in metastasization in MRK-003 treated mice. MK-0646 was not specific to hypoxic tumor environment, but substantially increased the median survival of treated mice compared to controls. NSCLC cells evaded MK-0646 treatment by activating EGF-R and ErbB3 exclusively both in vivo and in five cell lines in vitro. This phenomenon is achieved at the level of protein stability. One outcome of MK-0646 treatment is that cells poorly or not responsive to Erlotinib become more responsive to it. Sequential treatment with MK-0646 (three weeks), then combination of MK-0646 plus Erlotinib prolonged median survival of mice dramatically. If the two drugs where administered in combination from day one no survival advantage was observed in median survival compared to mice treated with Erlotinib alone and was actually less effective than MK-0646 used as single agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5227. doi:1538-7445.AM2012-5227

The tumor microenvironment (TME) of typical tumor types such as triple-negative breast cancer is featured by hypoxia and immunosuppression with abundant tumor-associated macrophages (TAMs), which also emerge as potential therapeutic targets for antitumor therapy. M1-like macrophage-derived exosomes (M1-Exos) have emerged as a promising tumor therapeutic candidate for their tumor-targeting and macrophage-polarization capabilities. However, the limited drug-loading efficiency and stability of M1-Exos have hindered their effectiveness in antitumor applications. Here, a hybrid nanovesicle is developed by integrating M1-Exos with AS1411 aptamer-conjugated liposomes (AApt-Lips), termed M1E/AALs. The obtained M1E/AALs are loaded withperfluorotributylamine (PFTBA)and IR780, as P-I, to construct P-I@M1E/AALs for reprogramming TME by alleviating tumor hypoxia and engineering TAMs. P-I@M1E/AAL-mediated tumor therapy enhances the in situ generation of reactive oxygen species, repolarizes TAMs toward an antitumor phenotype, and promotes the infiltration of T lymphocytes. The synergistic antitumor therapy based on P-I@M1E/AALs significantly suppresses tumor growth and prolongs the survival of 4T1-tumor-bearing mice. By integrating multiple treatment modalities, P-I@M1E/AAL nanoplatform demonstrates a promising therapeutic approach for overcoming hypoxic and immunosuppressive TME by targeted TAM reprogramming and enhanced tumor photodynamic immunotherapy. This study highlights an innovative TAM-engineering hybrid nanovesicle platform for the treatment of tumors characterized by hypoxic and immunosuppressive TME.

Colorectal cancer (CRC) remains a major global health challenge, particularly in advanced stages where drug resistance leads to high recurrence rates and poor survival outcomes. This study investigates a novel therapeutic approach combining gambogic acid (GA) with manganese dioxide (MnO2) nanoparticles (MBG NPs) to enhance anti-tumor efficacy in the acidic and hypoxic tumor microenvironment (TME). The development of MBG NPs involved conjugating MnO2 nanosheets with bovine serum albumin (BSA) for effective GA encapsulation, optimizing the delivery of both components. We explored the potential of Mn2+ ions released from MnO2 to synergize with GA to alleviate tumor hypoxia and modulate the TME, thereby improving immune response. In vitro assays demonstrated significant cytotoxicity of MBG NPs against mouse colon cancer cells (CT26 cells), with enhanced apoptosis and elevated reactive oxygen species (ROS) levels. In vivo studies using BALB/c mice showed that treatment with MBG NPs significantly reduced tumor volumes and improved survival rates compared to controls. Additionally, MBG NPs combined with programmed death-1 inhibitor (aPD-1) further augmented therapeutic effects. Histological analyses confirmed tumor necrosis and changes in TME composition, indicating the potential of this synergistic strategy to overcome drug resistance in microsatellite stable (MSS) CRC, inhibit tumor growth and benefit patient survival. These findings highlight the promising application of nanoparticle-based platforms in enhancing immunotherapy outcomes for advanced colorectal cancer.

Organic Sonosensitizers-based SDT with enhanced ROS generation

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Ultrasound-responsive Pickering emulsions enable extracellular matrix disruption for enhanced sonodynamic immunotherapy.

Radiation therapy (RT) is a mainstream clinical approach in cancer treatment. However, the therapeutic efficacy of RT is greatly hindered by the presence of excessive hydrogen peroxide (H2O2) in the hypoxic region of the solid tumor, thus leading to tumor recurrence and metastasis. Herein, a thioketal-linked amphiphilic nano-assembly (MTS) loaded with hydrophobic manganese oxide (HMO) nanoparticles (MTS@HMO) is examined as a promising multi-purpose reactive oxygen species (ROS)-catalytic nanozyme for transforming an RT-resistant hypoxic tumor microenvironment (TME) into an RT-susceptible one by scavenging ROS in the hypoxic core of the solid tumor. After intravenous injection, the MTS@HMO nano-assembly was able to sense and be degraded by the abundant ROS in the hypoxic TME, thereby releasing HMO particles for subsequent scavenging of H2O2. The oxygen generated during peroxide scavenging then relieved the hypoxic TME, thereby resulting in an increased sensitivity of the hypoxic tumor tissue towards RT. Moreover, the in situ hypoxic status was monitored via the T1-enhanced magnetic resonance (MR) imaging of the Mn2+ ions generated by the ROS-mediated degradation of HMO. The in vitro results demonstrated a significant H2O2 elimination and enhanced oxygen generation after the treatment of the MTS@HMO nano-assembly with tumor cells under hypoxic conditions, compared to the control MTS group. In addition, the combination of RT and pre-treatment with MTS@HMO nano-assembly significantly amplified the permanent DNA strand breaks in tumor cells compared to the control RT group. More importantly, the in vivo results proved that the systemic injection of the MTS@HMO nano-assembly prior to RT irradiation enhanced the RT-mediated tumor suppression and down-regulated the hypoxic marker of HIF-1α in the solid tumor compared to the control RT group. Overall, the present work demonstrates the great potential of the versatile ROS-catalytic hypoxia modulating strategy using the MTS@HMO nano-assembly to enhance the RT-induced antitumor efficacy in hypoxic solid tumors.

Modulation of tumor hypoxia and redox microenvironment using nanomedicines for enhanced cancer photodynamic therapy

A clinically acceptable strategy for sensitizing anti-PD-1 treatment by hypoxia relief

Hypoxia, or lack of oxygen, is a key modulator of the tumor microenvironment and is associated with immunosuppression, invasiveness, angiogenesis, radiotherapy resistance and poor patient outcomes. Methods to increase oxygen in the hypoxic tumor niche have failed due to the long distance oxygen must diffuse from dysfunctional vasculature to the hypoxic cells. To address this problem, we engineered OMX oxygen carriers that bind oxygen with high affinity and can specifically deliver oxygen to severely hypoxic tumor regions without afecting normoxic tissues. Here, we show that OMX extravasates through leaky tumor vasculature, penetrates into and delivers oxygen to hypoxic tumor tissue. This reduces hypoxia in the tumor microenvironment and sensitizes tumors to therapy. Specifically, OMX accumulation in tumors leads to an increase in tumor oxygenation in mouse human xenograft and immunocompetent rat orthotopic models, as assessed by direct oxygen measurement (oxygen probe), by18F-FMISO PET in vivo imaging and by IHC and ELISA assessment of hypoxia markers (pimonidazole, CAIX, CCI, HIF-1). Moreover, OMX-mediated tumor oxygenation increases efficacy of radiation therapy as demonstrated by ex-vivo clonogenic assay and tumor growth delay. Importantly, while radiation alone only moderately delays tumor growth, combination of OMX and radiation therapy leads to tumor eradication in >50% of tumors. In addition to the enhancement of radiation therapy with OMX, we are currently exploring the capacity of OMX to ameliorate immunosuppressive microenvironment caused by hypoxia. Results from a Phase 0 clinical trial in canine cancer patients indicate that OMX is well tolerated and safe to use in combination with standard of care radiation therapy even in aged and fragile canine patient populations. Similar to rodent tumor models, OMX penetrates the tumor tissue in spontaneous canine brain and melanoma tumors. Furthermore, OMX accumulation correlates with reduced hypoxia in the tumor microenvironment as assessed by multiple hypoxia markers using quantitative IHC and ELISA. Taken together, these preclinical safety and efficacy data in both rodents and canines strongly support our IND submission to initiate human clinical trials in newly diagnosed glioblastoma patients. Results from completed and ongoing studies support the potential of OMX as modulator of hypoxic tumor microenvironment that may significantly impact the effectiveness of radiotherapy, chemotherapy and immunotherapy in a variety of solid tumors where hypoxia is major contributor to therapeutic resistance. Citation Format: Ana Krtolica, Natacha Le Moan, Philberta Leung, Youngho Seo, Jonathan Winger, Henry Van Brocklin, Michael Kent, Stephen Cary. Sensitizing the hypoxic tumor microenvironment with OMX, a breakthrough oxygen delivery protein: From protein engineering to clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2790.

Uveal melanoma (UM) is a leading intraocular malignancy with a high 5-year mortality rate, and radiotherapy is the primary approach for UM treatment. However, the elevated lactic acid, deficiency in ROS, and hypoxic tumor microenvironment have severely reduced the radiotherapy outcomes. Hence, this study devised a novel CoMnFe-layered double oxides (LDO) nanosheet with multienzyme activities for UM radiotherapy enhancement. On one hand, LDO nanozyme can catalyze hydrogen peroxide (H2O2) in the tumor microenvironment into oxygen and reactive oxygen species (ROS), significantly boosting ROS production during radiotherapy. Simultaneously, LDO efficiently scavenged lactic acid, thereby impeding the DNA and protein repair in tumor cells to synergistically enhance the effect of radiotherapy. Moreover, density functional theory (DFT) calculations decoded the transformation pathway from lactic to pyruvic acid, elucidating a previously unexplored facet of nanozyme activity. The introduction of this innovative nanomaterial paves the way for a novel, targeted, and highly effective therapeutic approach, offering new avenues for the management of UM and other cancer types.

Exosome membrane-sheathed and multi-stimuli-responsive MnO2 nanoparticles with self-oxygenation and energy depletion abilities potentiate the sonodynamic therapy of hypoxic tumors

As prospective phototheranostic agents for cancer imaging and therapy, semiconducting organic molecule-based nanomedicines are developed. However, near-infrared (NIR) emission, and tunable type I (O2 • -) and type II (1O2) photoinduced reactive oxygen species (ROS) generation to boost cancer photoimmunotherapy remains a big challenge. Herein, a series of D-π-A structures, NIR absorbing perylene diimides (PDIs) with heavy atom bromide modification at the bay position of PDIs are prepared for investigating the optimal photoinduced type I/II ROS generation. The heavy atom effect has demonstrated a reduction of molecular ∆EST and promotion of the intersystem crossing processes of PDIs, enhancing the photodynamic therapy (PDT) efficacy. The modification of three bromides and one pyrrolidine at the bay position of PDI (TBDT) has demonstrated the best type I/II PDT performance by batch experiments and theoretical calculations. TBDT based nanoplatforms (TBDT NPs) enable type I/II PDT in the hypoxic tumor microenvironment as a strong immunogenic cell death (ICD) inducer. Moreover, TBDT NPs showing NIR emission allow in vivo bioimaging guided phototherapy of tumor. This work uses novel PDIs with adjustable type I/II ROS production to promote antitumor immune response and accomplish effective tumor eradication, consequently offering molecular guidelines for building high-efficiency ICD inducers.

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.