Mouse Breast Tumor Model Research Articles

Ketamine promotes breast tumor growth in a mouse breast tumor model involving with high expression of miR-27b-3p and EGFR.

Non-medical use of ketamine as an adulterant to ecstasy is more prevalent than amphetamine in Taiwan. Ketamine's effect on immunosuppression might play some functional role in tumor growth, while it is still controversial whether ketamine abuse could increase tumor growth or not. This study aimed to investigate the influence of ketamine addiction in breast tumors and related gene expressions. The effect of ketamine treatment on proliferation, colony formation, migration, and invasion of triple-negative breast cancer cell line EO771 was examined. In addition, a ketamine addiction mice model was established by intraperitoneal injection (IP) of ketamine in mice and used to investigate the effects of ketamine addiction on tumor growth and the possible mechanisms. In the in vitro studies, ketamine treatment at different concentrations did not affect EO771 cell proliferation and colony formation. But ketamine did enhance migration and invasion of EO771 cells. The in vivo experiments showed significantly increased breast tumor volume and weight in ketamine-addicted mice than in normal saline groups. miR-27b-3p level, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) significantly increased in tumors of ketamine addiction mice compared to control mice. In vivo evidence showed that Ketamine might increase tumor growth on the tumor microenvironment, and miR-27b-3p, HER2, and EGFR might play a role in the process.

Vitexin alleviates breast tumor in mice via skewing TAMs toward an iNOS+ profile orchestrating effective CD8+ T cell activation

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) and often induce an immunosuppressed state. Vitexin is an apigenin flavone glycoside found in food and medicinal plants. Vitexin inhibits the growth of many tumors including breast cancer. However, the exact mechanism of the immune response of vitexin in breast cancer growth is not known. In this study, we investigated the potential anti-tumor mechanism of vitexin on regulating TAMs function in vivo and in vitro. An allograft breast tumor mouse model was established by injecting mouse 4T1 cells into female BALB/c mice, and the effect of vitexin on modulating TAMs and CD8+ T cells was assessed. Then, a cell model was used to illustrate the polarization effect of vitexin on the phenotype of macrophages. Finally, an inducible nitric oxide synthase (iNOS) inhibitor, 1400 W, was used to verify the effective target of vitexin. In vivo studies showed that vitexin programs TAMs to an iNOS+ profile and activates CD8+ T cells. In vitro experiments showed that vitexin, together with lipopolysaccharide and interferon-gamma, polarises macrophages to an M1 profile. Mechanistic validation studies showed that the antitumor effect of vitexin was partially attenuated by reduced iNOS expression, but still had an effect on CD8+ T cells in the spleen and blood.

A bio-responsive, cargo-catchable gel for postsurgical tumor treatment via ICD-based immunotherapy.

Tumor recurrence and metastasis after surgery remain challenges for tumor treatment. Strategy that can promote the immunogenicity, activate adaptative immune response and eliminate post-operative immunosuppression would be a promising way to achieve a desired clinical benefit. In this study, immunogenic cell death (ICD) priming anti-tumor adaptive immune response was executed to potentiate immune checkpoint blockade (ICB) therapy through the PD1/PDL1 pathway for postsurgical treatment. Here, we present a bio-responsive and cargo-catchable gel depot composed of pullulan and chitosan cross-linking through matrix metalloproteinase (MMP) sensitive peptide for co-delivery of anti-programmed death-ligand 1 antibody (aPDL1) and doxorubicin -encapsulated liposomes (DOX-Lip). This drug carrier showed expected ability to respond to the highly expressed MMP in postsurgical tumor microenvironment (TME). In vivo studies on 4T1 breast tumor mouse model demonstrated that the gel depot could efficiently prolong the mouse survival after tumor resection by preventing tumor recurrence and metastasis. The results suggested that ICD combining with PD1/PDL1 blockade based on the bio-responsive and cargo-catchable gel depot could facilitate the maturation of DCs and reverse the immunosuppressive environment in tumor resection area, thus amplifying the systemic anti-tumor immune response.

Bifunctional Fusion Membrane‐Based Hydrogel Enhances Antitumor Potency of Autologous Cancer Vaccines by Activating Dendritic Cells

AbstractAutologous tumor cell vaccine is an individualized tumor treatment strategy to elicit tumor‐specific immune responses. However, it is difficult to exert efficacy in “cold” tumors, which lack tumor‐infiltrating T cells and are not sensitive to immunotherapy. Here, a dendritic cells (DCs) activation hydrogel is constructed based on bifunctional fusion membrane nanoparticles (FM‐NPs) composed of autologous tumor cell membranes and Mycobacterium phlei membrane extracts. The FM‐NPs not only provide tumor antigens but also can activate DCs. Afterward, the FM‐NPs are loaded in an injectable alginate hydrogel together with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF releases rapidly and recruits DCs to hydrogel, while FM‐NPs retain in hydrogel to continuously interconnected with DCs, enriching mature DCs loaded with tumor antigens. In the 4T1 mouse breast tumor model, this hydrogel promotes the maturation of DCs in tumor‐draining lymph nodes and induces sufficient effector memory T cells that migrate to the tumor microenvironment. This successfully converts the “cold” tumors to “hot”, thus exerting a significant antitumor effect synergized with the PD‐1 antibody. It is also verified the inhibitory effect of FM‐NPs using human gastric cancer organoids. Altogether, these findings demonstrate the application potential for the bifunctional fusion membrane‐based hydrogel as an autologous tumor vaccine by activating DCs.

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8+ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4+ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

IntroductionCD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation.MethodsCD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model.ResultsSHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts.ConclusionOur findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.

Bacterial cytoplasmic membranes synergistically enhance the antitumor activity of autologous cancer vaccines.

Cancer vaccines based on resected tumors from patients have gained great interest as an individualized cancer treatment strategy. However, eliciting a robust therapeutic effect with personalized vaccines remains a challenge because of the weak immunogenicity of autologous tumor antigens. Utilizing exogenous prokaryotic constituents that act as adjuvants to enhance immunogenicity is a promising strategy to overcome this limitation. However, nonspecific stimulation of the immune system may elicit an undesirable immunopathological state. To specifically trigger sufficient antitumor reactivity without notable adverse effects, we developed an antigen and adjuvant codelivery nanoparticle vaccine based on Escherichia coli cytoplasmic membranes (EMs) and tumor cell membranes (TMs) from resected autologous tumor tissue. Introduction of the EM into the hybrid membrane nanoparticle vaccines (HM-NPs) induced dendritic cell maturation, thus activating splenic T cells. HM-NPs showed efficacy in immunogenic CT26 colon and 4T1 breast tumor mouse models and also efficiently induced tumor regression in B16-F10 melanoma and EMT6 breast tumor mouse models. Furthermore, HM-NPs provoked a strong tumor-specific immune response, which not only extended postoperative animal survival but also conferred long-term protection (up to 3 months) against tumor rechallenge in a CT26 colon tumor mouse model. Specific depletion of different immune cell populations revealed that CD8+ T and NK cells were crucial to the vaccine-elicited tumor regression. Individualized autologous tumor antigen vaccines based on effective activation of the innate immune system by bacterial cytoplasmic membranes hold great potential for personalized treatment of postoperative patients with cancer.

Abstract 1804: Modulation of Nr2f2 reprograms tumor blood vessels to enhance anti-tumor immunity and immunotherapy

Abstract A critical limitation to cancer immunotherapy is posed by the paucity of anti-tumor T cells in tumor tissues, even after therapeutic intervention. Development of effective strategies to enhance T cell recruitment remains challenging. As a critical regulator of immune cell trafficking, the tumor vasculature is a promising target that can be utilized to enhance anti-tumor immunity. Functional vasculature requires specialization of endothelial cells (ECs): capillary ECs transport oxygen and nutrients to promote tumor growth, while venular ECs regulate the recruitment and extravasation of immune cells. The majority of the current vessel-targeting therapies are designed to deplete endothelial cells indiscriminately with the goal of starving tumor cells. Loss of venules as a consequence could further block sustained recruitment of anti-tumor T cells. An alternative strategy that instead promotes capillary-to-venule “reprogramming” could simultaneously inhibit the tumor-promoting activities of capillaries and preserve the T cell-recruiting functions of tumor vessels, and thus better augment anti-tumor immunity and benefit immunotherapies. Through single cell transcriptomic profiling of tumor ECs, we found the nuclear receptor Nr2f2 to be specifically expressed in tumor-associated venules but not in capillaries. Upon ectopic expression of Nr2f2 in tumor ECs, capillary vessels undergo fate switch and adopt the venular phenotype and functions, as confirmed by immune profiling and single cell transcriptomic analyses. Nr2f2-driven capillary-to-venule reprogramming enhanced the recruitment of cytotoxic T cells into the tumor tissue, and led to a significant reduction of tumor burden in mouse breast and pancreatic tumor models. Interestingly, Nr2f2-driven EC fate switch enhanced the anti-tumor efficacy of immunotherapies, including immune checkpoint blockade and adoptive cell therapy by transferring T cells specific for a surrogate tumor cell antigen. Collectively, these data suggest that strategies that reprogram tumor vessels from capillaries into venules can be exploited to enhance anti-tumor immune responses and sensitize tumors to immunotherapy, especially in immunologically cold tumors. Citation Format: Yu Zhu, Nicole Lazarus, Kevin Brulois, Nicole Salazar, Theresa Dinh, Junliang Pan, Eugene Butcher. Modulation of Nr2f2 reprograms tumor blood vessels to enhance anti-tumor immunity and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1804.

Abstract PO022: Metabolic and transcriptional adaptations under hypoxic stress promote breast cancer progression

Abstract To sustain energy production for survival in hypoxia, tumor cells undergo metabolic rewiring predominantly by accentuating glucose uptake through hypoxia-driven enhanced expression of glycolytic enzymes. 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is one of the hypoxia response glycolytic enzyme which primarily balances the glucose flux between glycolysis and pentose phosphate pathway. However, the necessity to increase PFKFB4 expression under hypoxia is poorly understood. Here, we show that PFKFB4 dependent adaptation in hypoxia promotes a tumor-intrinsic genetic alteration acquiring invasive phenotype in breast cancer. Specifically, we found that CRISPR-Cas9 PFKFB4-knock-out (KO) breast cancer cells were defective in migration under normoxic condition (21% O2) compared to wild-type (WT) cells; while this migratory defect was found more severe under hypoxic stress (1% O2). To elucidate the mechanisms of PFKFB4-driven migration, we performed RNA-sequencing which revealed that PFKFB4 induces expression of focal adhesion pathway genes including ITGB3 (integrin β3), which are essential regulators of migration and invasion. While validating the target genes by qPCR and immunoblotting, we found that a great number of focal adhesion target genes were hypoxia-response genes including ITGB3, as their expression was significantly enhanced in hypoxia, which was completely abrogated in the absence of PFKFB4. Rescuing ITGB3-expression in PFKFB4-KO cells was sufficient to restore the migratory defects in both normoxic and hypoxic conditions, suggesting that PFKFB4 promotes migration by inducing focal adhesion genes such as ITGB3 expression. Previously we have shown that PFKFB4 directly activates steroid receptor coactivator 3 (SRC-3, also known as NCOA3) so we analyzed promoter occupancy of SRC-3 on the proximal promoter of ITGB3. Our data revealed strong occupancy of SRC-3 on the ITGB3 promoter, which also contains a canonical hypoxia response element (HRE), suggesting that PFKFB4/SRC-3 signaling axis may stimulate HIF-1A transcriptional response. By further elucidating the roles of PFKFB4/SRC-3/HIF-1A axis in hypoxia in breast tumor mouse models, we will demonstrate how metabolic and transcriptional adaptations in the TME stimulates breast cancer progression and metastasis. Citation Format: Tao Dai, Aaron Novickis, Eriko Katsuta, Kazuaki Takabe, Scott I. Abrams, Subhamoy Dasgupta. Metabolic and transcriptional adaptations under hypoxic stress promote breast cancer progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO022.

Enzyme-instructed hybrid nanogel/nanofiber oligopeptide hydrogel for localized protein delivery

Enzyme-catalysis self-assembled oligopeptide hydrogel holds great interest in drug delivery, which has merits of biocompatibility, biodegradability and mild gelation conditions. However, its application for protein delivery is greatly limited by inevitable degradation of enzyme on the encapsulated proteins leading to loss of protein activity. Moreover, for the intracellularly acted proteins, cell membrane as a primary barrier hinders the transmembrane delivery of proteins. The internalized proteins also suffer from acidic and enzymatic degradation in endosomes and lysosomes. We herein develop a protease-manipulated hybrid nanogel/nanofiber hydrogel for localized delivery of intracellularly acted proteins. The embedded polymeric nanogels (CytoC/aNGs) preserve activity of cytochrome c (CytoC) that is an intracellular activator for cell apoptosis as a model protein against proteolysis, and do not affect the gelation properties of the protease-catalysis assembled hydrogels. The injectable hydrogel (CytoC/aNGs/Gel) serves as a reservoir to enhance intratumoral retention and realize sustainable release of CytoC/aNGs. The released CytoC/aNGs increase cellular uptake of CytoC and enhance its intracellular delivery to its target site, cytoplasm, resulting in favorable apoptosis-inducing and cytotoxic effects. We show that a single local administration of CytoC/aNGs/Gel efficiently inhibit the tumor growth in the breast tumor mouse model.

Synergistic breast cancer suppression efficacy of doxorubicin by combination with glycyrrhetinic acid as an angiogenesis inhibitor

BackgroundTherapeutic regimens of breast cancer treatment are increasingly inclined to adopt combination strategy based on the broad spectrum antitumor effect of doxorubicin (Dox). Currently, combination therapy comprises of conventional anti-cancer drugs and angiogenesis inhibitors have been corroborated as an effective approach in cancer treatment. PurposeWe explored the ability of a natural anti-angiogenic compound glycyrrhetinic acid (GA), derived from an edible-medicinal herb licorice, to enhance the breast cancer suppression effect of Dox. Study designThe drug ratio of GA and Dox with synergistic anticancer effect against MCF-7 cells was optimized by combination index (CI) value in vitro, followed by evaluation of the improved anticancer effects and reduced side-effects of this combination in vitro and in vivo. MethodsCell viability was measured by MTT assay. Analyses of mitochondrial membrane potential and cell apoptosis on MCF-7 cells were performed by JC-1 dye and Annexin V-FITC/PI assays. The cellular accumulation of Dox when combined with GA was evaluated. Levels of apoptosis-related proteins in MCF-7 cells were measured by Western blot analysis. Synergistic anti-angiogenic effects on HUVECs were evaluated. A breast cancer mouse model was established to investigate the anti-tumor effects in vivo. ResultsBased on the optimization by CI value, Dox and GA at 1:20 molar ratio was chosen as the optimal combination drug ratio that exhibited synergistic effect against MCF-7 breast cancer cells. In addition, the combination of GA and Dox exhibited significantly enhanced cytotoxicity, apoptosis, and loss of mitochondrial membrane potential via the upregulation of a mitochondrial-dependent apoptosis pathway against MCF-7 cells. Interestingly, the addition of GA increased the intracellular accumulation of Dox in MCF-7 cells. Moreover, VEGF-induced HUVECs proliferation, migration, and tube formation were strongly inhibited by Dox when used with GA via the significant down-regulation of VEGFR2-mediated pathway, indicating that the combination of Dox and GA could exhibit ideal synergistic anti-angiogenesis effect. Expectedly, the enhanced anti-tumor efficacy of Dox and reduced Dox-induced cardiotoxicity when used in combination with GA were evident in a mouse breast tumor model. ConclusionsThese findings support that the combination of Dox with GA is a novel and promising therapeutic strategy for the treatment of breast cancer.

Intratumoral Anti-CTLA-4 Blockade Can Potentiate Radiation-Induced Anti-Tumor Immunity In Breast Cancer

Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with solid malignancies. However, the number of responders remain low. We have previously shown in the 4T1 mouse breast tumor model that resistance to anti-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Cancer Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells (Matsumura et al J Immunol 2008; Ruocco et al J Clin Invest 2012). Our prior work, done exclusively via systemic intraperitoneal CTLA-4 treatment, support the hypothesis that RT generates an in situ vaccine while CTLA-4 expands primed T cells. Here we test whether intratumoral delivery can elicit equally robust responses when combined with fractionated RT. BALB/c mice were inoculated s.c. with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 6 treatment groups (N = 6-8 mice/group): control, RT alone, i.p. anti-CTLA-4 alone, i.t. anti-CTLA-4 alone, RT+ i.p. anti-CTLA-4 or RT+ i.t. anti-CTLA-4. RT was given in 3 fractions of 8 Gy on days 13,14 and 15 post-tumor inoculation. Anti-CTLA-4 mAb (Clone 9H10, 200 ug/mouse) was given on days 15, 18 and 21. Animals were followed for tumor growth and survival. In parallel experiments (N = 5 mice/group), mice were euthanized on day 22 for immunophenotyping of tumor-infiltrating lymphocytes and ex vivo IFNg production by tumor-specific CD8 T-cells in the draining lymph nodes. Treatment of 4T1 tumor-bearing mice with anti-CTLA-4 checkpoint blockade alone, whether administered intraperitoneal or intratumoral, was unable to exert any local control on tumor progression nor improve overall survival. As expected, focal RT significantly delayed tumor growth (Control vs RT, p<0.05) but had marginal effects on survival (median 35 days in control vs 40 days in RT grp, p = 0.301). The combination of RT and CTLA-4 blockade elicited the best responses, with i.p.-treated mice displaying a slight advantage vs i.p.-treated in both tumor inhibition (RT+i.t. CTLA-4 vs RT+i.p. CTLA4, p<0.05) and median survival (44.5 days in RT+i.t. CTLA-4 vs 42 days RT+i.p. CTLA4). Overall these results support rational combination of intratumoral anti-CTLA-4 with local RT. Ongoing work will further optimize the therapeutic response to combination treatment, including the addition of PD-1 blockade.

Promising potential of [177Lu]Lu-DOTA-folate to enhance tumor response to immunotherapy\u2014a preclinical study using a syngeneic breast cancer model

PurposeIt was previously demonstrated that radiation effects can enhance the therapy outcome of immune checkpoint inhibitors. In this study, a syngeneic breast tumor mouse model was used to investigate the effect of [177Lu]Lu-DOTA-folate as an immune stimulus to enhance anti-CTLA-4 immunotherapy.MethodsIn vitro and in vivo studies were performed to characterize NF9006 breast tumor cells with regard to folate receptor (FR) expression and the possibility of tumor targeting using [177Lu]Lu-DOTA-folate. A preclinical therapy study was performed over 70 days with NF9006 tumor-bearing mice that received vehicle only (group A); [177Lu]Lu-DOTA-folate (5 MBq; 3.5 Gy absorbed tumor dose; group B); anti-CTLA-4 antibody (3 × 200 μg; group C), or both agents (group D). The mice were monitored regarding tumor growth over time and signs indicating adverse events of the treatment.Results[177Lu]Lu-DOTA-folate bound specifically to NF9006 tumor cells and tissue in vitro and accumulated in NF9006 tumors in vivo. The treatment with [177Lu]Lu-DOTA-folate or an anti-CTLA-4 antibody had only a minor effect on NF9006 tumor growth and did not substantially increase the median survival time of mice (23 day and 19 days, respectively) as compared with untreated controls (12 days). [177Lu]Lu-DOTA-folate sensitized, however, the tumors to anti-CTLA-4 immunotherapy, which became obvious by reduced tumor growth and, hence, a significantly improved median survival time of mice (> 70 days). No obvious signs of adverse effects were observed in treated mice as compared with untreated controls.ConclusionApplication of [177Lu]Lu-DOTA-folate had a positive effect on the therapy outcome of anti-CTLA-4 immunotherapy. The results of this study may open new perspectives for future clinical translation of folate radioconjugates.

Doxorubicin-Loaded Physalis Mottle Virus Particles Function as a pH-Responsive Prodrug Enabling Cancer Therapy.

The controlled release of drugs using nanoparticle-based delivery vehicles is a promising strategy to improve the safety and efficacy of chemotherapy. A simple, scalable, and reproducible strategy is developed to synthesize a drug delivery system (DDS) by loading 6-maleimidocaproyl-hydrazone doxorubicin (DOX-EMCH) into the empty core of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) via a combination of chemical conjugation to cysteine residues and π-π stacking interactions with the anchored doxorubicin molecule. The DOX-EMCH prodrug features an acid-sensitive hydrazine linker that triggers the release of doxorubicin in the slightly acidic extracellular tumor microenvironment or acidic endosomal or lysosomal compartments following cellular uptake. The VLP external surface is coated with polyethylene glycol (PEG) to prevent non-specific uptake and improve biocompatibility. The DOX-PhMV-PEG particles are stable in vitro and show greater efficacy in vivo compared to free doxorubicin in a breast tumor mouse model (using MDA-MB-231 cells and nude mice): 92% of the tumor-bearing mice treated with DOX-PhMV-PEG are completely cured compared to 27% of those treated with free doxorubicin under the same conditions, representing a 3.4-fold improvement. These results lay a foundation for the further development of this biological drug delivery system for a new generation of chemotherapy products.

Chitosan/γ-PGA nanoparticles-based immunotherapy as adjuvant to radiotherapy in breast cancer

Radiotherapy (RT) is an essential treatment modality for several types of cancer. Despite its therapeutic potential, RT is frequently insufficient to overcome the immunosuppressive nature of the tumor microenvironment, failing to control tumor metastases. Innovative immunomodulatory strategies, like immunostimulatory biomaterials could be used to boost the immunogenic effects of RT. Herein, we addressed the synergistic potential of immunostimulatory chitosan/poly(γ-glutamic acid) nanoparticles (Ch/γ-PGA NPs) combined with RT to induce antitumor immunity in the 4T1 orthotopic breast tumor mouse model. Non-treated animals had progressive primary tumor growth and developed splenomegaly and lung metastases. While RT decreased primary tumor burden, Ch/γ-PGA NPs-treatment decreased systemic immunosuppression and lung metastases. The combination therapy (RT + Ch/γ-PGA NPs) synergistically impaired 4T1 tumor progression, which was associated with a significant primary tumor growth and splenomegaly reduction, a decrease in the percentage of splenic immunosuppressive myeloid cells and an increase in antitumoral CD4+IFN-γ+ population. Notably, animals from the combination therapy presented less and smaller lung metastatic foci and lower levels of the systemic pro-tumor cytokines IL-3, IL-4, IL-10, and of the CCL4 chemokine, in comparison to non-treated animals. Overall, these results evidenced that Ch/γ-PGA NPs potentiate and synergize with RT, headlining their promising role as adjuvant anticancer strategies.

Tumor Cell Membrane‐Coated Liquid Metal Nanovaccine for Tumor Preventionǂ

Summary of main observation and conclusionHerein, we report on a tumor nanovaccine LMNP@CM that enhances the process of antigen‐presenting by stimulating the immune system to uptake tumor antigens actively. The nanovaccine is comprised of polyethylene glycol modified liquid metal nanoparticles (LMNP) and tumor cell membranes (CM) as antigens. Under 808 nm irradiation, the photothermal conversion effect of injected LMNP can cause mild local inflammation, and subsequently induces antigen‐presenting cells active recruitment and facilitates cellular uptake of tumor antigens. Meanwhile, because of the immune adjuvant effect of metal materials, the nanovaccine LMNP@CM promotes the maturation and activation of antigen‐presenting cells and induces anti‐tumor immune response effectively. By priming the host immune recognition of tumor antigens, this nanovaccine displays prophylactic effects and significantly suppresses tumor growth in a mouse breast tumor model. The nanovaccine LMNP@CM represents a novel strategy of utilizing light‐controlled means to actively induce anti‐tumor immune processes, showing advanced therapeutic potentials and robust adaptability for treating multiple tumors by changing the loaded antigens.

Bioreductively Activatable Prodrug Conjugates of Combretastatin A-1 and Combretastatin A-4 as Anticancer Agents Targeted toward Tumor-Associated Hypoxia.

The natural products combretastatin A-1 (CA1) and combretastatin A-4 (CA4) function as potent inhibitors of tubulin polymerization and as selective vascular disrupting agents (VDAs) in tumors. Bioreductively activatable prodrug conjugates (BAPCs) can enhance selectivity by serving as substrates for reductase enzymes specifically in hypoxic regions of tumors. A series of CA1-BAPCs incorporating nor-methyl, mono-methyl, and gem-dimethyl nitrothiophene triggers were synthesized together with corresponding CA4-BAPCs, previously reported by Davis (Mol. Cancer Ther. 2006, 5 (11), 2886), for comparison. The CA4-gem-dimethylnitrothiophene BAPC 45 proved exemplary in comparison to its nor-methyl 43 and mono-methyl 44 congeners. It was stable in phosphate buffer (pH 7.4, 24 h), was cleaved (25%, 90 min) by NADPH-cytochrome P450 oxidoreductase (POR), was inactive (desirable prodrug attribute) as an inhibitor of tubulin polymerization (IC50 > 20 μM), and demonstrated hypoxia-selective activation in the A549 cell line [hypoxia cytotoxicity ratio (HCR) = 41.5]. The related CA1-gem-dimethylnitrothiophene BAPC 41 was also promising (HCR = 12.5) with complete cleavage (90 min) upon treatment with POR. In a preliminary in vivo dynamic bioluminescence imaging study, BAPC 45 (180 mg/kg, ip) induced a decrease (within 4 h) in light emission in a 4T1 syngeneic mouse breast tumor model, implying activation and vascular disruption.

TRNA Queuosine Modification Enzyme Modulates the Growth and Microbiome Recruitment to Breast Tumors.

Background: Transfer RNA (tRNA) queuosine (Q)-modifications occur specifically in 4 cellular tRNAs at the wobble anticodon position. tRNA Q-modification in human cells depends on the gut microbiome because the microbiome product queuine is required for its installation by the enzyme Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) encoded in the human genome. Queuine is a micronutrient from diet and microbiome. Although tRNA Q-modification has been studied for a long time regarding its properties in decoding and tRNA fragment generation, how QTRT1 affects tumorigenesis and the microbiome is still poorly understood. Results: We generated single clones of QTRT1-knockout breast cancer MCF7 cells using Double Nickase Plasmid. We also established a QTRT1-knockdown breast MDA-MB-231 cell line. The impacts of QTRT1 deletion or reduction on cell proliferation and migration in vitro were evaluated using cell culture, while the regulations on tumor growth in vivo were evaluated using a xenograft BALB/c nude mouse model. We found that QTRT1 deficiency in human breast cancer cells could change the functions of regulation genes, which are critical in cell proliferation, tight junction formation, and migration in human breast cancer cells in vitro and a breast tumor mouse model in vivo. We identified that several core bacteria, such as Lachnospiraceae, Lactobacillus, and Alistipes, were markedly changed in mice post injection with breast cancer cells. The relative abundance of bacteria in tumors induced from wildtype cells was significantly higher than those of QTRT1 deficiency cells. Conclusions: Our results demonstrate that the QTRT1 gene and tRNA Q-modification altered cell proliferation, junctions, and microbiome in tumors and the intestine, thus playing a critical role in breast cancer development.

Tumor-Adhesive and pH-Degradable Microgels by Microfluidics and Photo-Cross-Linking for Efficient Antiangiogenesis and Enhanced Cancer Chemotherapy

Tumor angiogenesis with the vascular network formation provides nutrition and oxygen for cancer cells, promoting the proliferation and metastasis of malignant tumors. Bevacizumab (Bev) as an efficient antiangiogenic antibody is able to normalize the tumor vasculature with better blood flow and reduced interstitial fluid pressure, allowing drugs with more uniform distribution and deeper penetration into the tumor; however, it is highly difficult to realize the simultaneous delivery of Bev and anticancer drugs localized at the tumor tissue. Here, we prepared tumor-adhesive and pH-degradable poly(vinyl alcohol) (PVA) microgels for tumor-localized delivery of Bev and docetaxel (DTX), to achieve efficient antiangiogenesis and enhanced cancer chemotherapy. PVA microgels (∼200 μm) decorated with tissue-adhesive dopamine (DA) moieties were fabricated by a combination of high-throughput microfluidics technology and photo-cross-linking chemistry with a considerable coencapsulation efficiency for Bev and DTX. PVA microgels exhibited sustained drug release at the tumoral acidic conditions as the microgel degradation, and DA moieties on the microgels facilitated Bev with long retention at the tumor tissue, highly blocking the vascular endothelial growth factor (VEGF) and inhibiting tumor angiogenesis, as compared to free Bev or no DA-decorated microgels. In addition, the antitumor activity on the 4T1-Luc breast tumor mouse model treated with Bev/DTX-coloaded microgels showed obviously superior tumor growth inhibition than the other treatment groups, in which the combinational therapy efficacy of Bev and DTX mediated by the tumor-adhesive microgels was further confirmed by the immunohistochemistry (IHC) analysis. These PVA microgels with efficient antiangiogenesis and enhanced cancer chemotherapy provide a highly potential platform to treat different malignant tumors as well as the recurrent and metastatic tumors.

BHQ-Cyanine-Based "Off-On" Long-Circulating Assembly as a Ferroptosis Amplifier for Cancer Treatment: A Lipid-Peroxidation Burst Device.

Ferroptosis is an iron-dependent cell death caused by accumulation of lipid peroxidation (LPO), which is a new strategy for cancer treatment. Th current ferroptosis therapy nanodevices have low efficiency and side effects generally. Hence, we developed a Black Hole Quencher (BHQ)-based fluorescence "off-on" nanophotosensitizer complex assembly (CSO-BHQ-IR780-Hex/MIONPs/Sor). CSO-connected BHQ-IR780-Hex and -loaded magnetic iron oxide nanoparticles (MIONPs) and sorafenib (Sor) formed a very concise functionalized delivery system. CSO-BHQ-IR780-Hex disassembled by GSH attack and released IR780-Hex, MIONPs, and sorafenib. IR780-Hex anchored to the mitochondrial membrane, which would contribute to amplifying the efficiency of the photosensitizer. When NIR irradiation was given to CSO-BHQ-IR780-Hex/MIONPs/Sor-treated cells, iron supply increased, the xCT/GSH/GPX-4 system was triggered, and a lot of LPO burst. A malondialdehyde test showed that LPO in complex assembly-treated cells was explosive and increased about 18-fold compared to the control. The accumulation process of particles was monitored by an IR780-Hex photosensitizer, which showed an excellent tumor target ability by magnetic of nanodevice in vivo. Interestingly, the half-life of sorafenib in a nanodevice was increased about 26-fold compared to the control group. Importantly, the complex assembly effectively inhibits tumor growth in the breast tumor mouse model. This work would provide ideas in designing nanomedicines for the ferroptosis treatment of cancer.