Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

Suppression of breast cancer-associated bone loss with osteoblast proteomes via Hsp90ab1/moesin-mediated inhibition of TGFß/FN1/CD44 signaling.

Background: Bone is a frequent site of metastases from breast cancer, but existing therapeutic options are not satisfactory. Although osteoblasts have active roles in cancer progression by assisting the vicious bone-destructive cycle, we employed a counterintuitive approach of activating pro-tumorigenic Wnt signaling and examined the paradoxical possibility of developing osteoblast-derived tumor-suppressive, bone-protective secretomes. Methods: Wnt signaling was activated by the overexpression of Lrp5 and ß-catenin in osteoblasts as well as a pharmacological agent (BML284), and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of osteolysis. To explore the unconventional regulatory mechanism of the action of Wnt-activated osteoblasts, whole-genome proteomics analysis was conducted, followed by immunoprecipitation and gain- and loss-of-function assays. Results: While osteoblasts did not present any innate tumor-suppressing ability, we observed that the overexpression of Lrp5 and ß-catenin in Wnt signaling made their CM tumor-suppressive and bone-protective. The growth of breast cancer cells and tissues was inhibited by Lrp5-overexpressing CM (Lrp5 CM), which suppressed mammary tumors and tumor-driven bone destruction in a mouse model. Lrp5 CM also inhibited the differentiation and maturation of bone-resorbing osteoclasts by downregulating NFATc1 and cathepsin K. The overexpression of Lrp5 upregulated osteopontin that enriched Hsp90ab1 (Hsp90 beta) and moesin (MSN) in Lrp5 CM. Hsp90ab1 and MSN are atypical tumor-suppressing proteins since they are multi-tasking, moonlighting proteins that promote tumorigenesis in tumor cells. Importantly, Hsp90ab1 immuno-precipitated latent TGFß and inactivated TGFß, whereas MSN interacted with CD44, a cancer stem-cell marker, as well as fibronectin 1, an ECM protein. Furthermore, Hsp90ab1 and MSN downregulated KDM3A that demethylated histones, together with PDL1 that inhibited immune responses. Conclusion: In contrast to inducing tumor-enhancing secretomes and chemoresistance in general by inhibiting varying oncogenic pathways in chemotherapy, this study presented the unexpected outcome of generation tumor-suppressive secretomes by activating the pro-tumorigenic Wnt pathway. The results shed light on the contrasting role of oncogenic signaling in tumor cells and osteoblast-derived secretomes, suggesting a counterintuitive option for the treatment of breast cancer-associated bone metastasis.

Pharmacological Activation of cGAS for Cancer Immunotherapy.

When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.

Immunoprotective effect of an in silico designed multiepitope cancer vaccine with BORIS cancer-testis antigen target in a murine mammary carcinoma model.

In our previous study, immunoinformatic tools were used to design a novel multiepitope cancer vaccine based on the most immunodominant regions of BORIS cancer-testis antigen. The final vaccine construct was an immunogenic, non-allergenic, and stable protein consisted of multiple cytotoxic T lymphocytes epitopes, IFN-γ inducing epitopes, and B cell epitopes according to bioinformatic analyzes. Herein, the DNA sequence of the final vaccine construct was placed into the pcDNA3.1 vector as a DNA vaccine (pcDNA3.1-VAC). Also, the recombinant multiepitope peptide vaccine (MPV) was produced by a transfected BL21 E. coli strain using a recombinant pET-28a vector and then, purified and screened by Fast protein liquid chromatography technique (FPLC) and Western blot, respectively. The anti-tumor effects of prophylactic co-immunization with these DNA and protein cancer vaccines were evaluated in the metastatic non-immunogenic 4T1 mammary carcinoma in BALB/c mice. Co-immunization with the pcDNA3.1-VAC and MPV significantly (P < 0.001) increased the serum levels of the MPV-specific IgG total, IgG2a, and IgG1. The splenocytes of co-immunized mice exhibited a significantly higher efficacy to produce interleukin-4 and interferon-γ and proliferation in response to MPV in comparison with the control. The prophylactic co-immunization regime caused significant breast tumors' growth inhibition, tumors' weight decrease, inhibition of metastasis formation, and enlarging tumor-bearing mice survival time, without any considerable side effects. Taking together, this cancer vaccine can evoke strong immune response against breast tumor and inhibits its growth and metastasis.

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

Effect of immune modulation on the skeletal muscle mitochondrial exercise response: An exploratory study in mice with cancer.

Cancer causes mitochondrial alterations in skeletal muscle, which may progress to muscle wasting and, ultimately, to cancer cachexia. Understanding how exercise adaptations are altered by cancer and cancer treatment is important for the effective design of exercise interventions aimed at improving cancer outcomes. We conducted an exploratory study to investigate how tumor burden and cancer immunotherapy treatment (anti-PD-1) modify the skeletal muscle mitochondrial response to exercise training in mice with transplantable tumors (B16-F10 melanoma and EO771 breast cancer). Mice remained sedentary or were provided with running wheels for ~19 days immediately following tumor implant while receiving no treatment (Untreated), isotype control antibody (IgG2a) or anti-PD-1. Exercise and anti-PD-1 did not alter the growth rate of either tumor type, either alone or in combination therapy. Untreated mice with B16-F10 tumors showed increases in most measured markers of skeletal muscle mitochondrial content following exercise training, as did anti-PD-1-treated mice, suggesting increased mitochondrial content following exercise training in these groups. However, mice with B16-F10 tumors receiving the isotype control antibody did not exhibit increased skeletal muscle mitochondrial content following exercise. In untreated mice with EO771 tumors, only citrate synthase activity and complex IV activity were increased following exercise. In contrast, IgG2a and anti-PD-1-treated groups both showed robust increases in most measured markers following exercise. These results indicate that in mice with B16-F10 tumors, IgG2a administration prevents exercise adaptation of skeletal muscle mitochondria, but adaptation remains intact in mice receiving anti-PD-1. In mice with EO771 tumors, both IgG2a and anti-PD-1-treated mice show robust skeletal muscle mitochondrial exercise responses, while untreated mice do not. Taken together, we postulate that immune modulation may enhance skeletal muscle mitochondrial response to exercise in tumor-bearing mice, and suggest this as an exciting new avenue for future research in exercise oncology.

Abscopal Effect of Radiotherapy Enhanced with Immune Checkpoint Inhibitors of Triple Negative Breast Cancer in 4T1 Mammary Carcinoma Model.

Local radiotherapy (RT) is important to manage metastatic triple-negative breast cancer (TNBC). Although RT primarily reduces cancer cells locally, this control can be enhanced by triggering the immune system via immunotherapy. RT and immunotherapy may lead to an improved systemic effect, known as the abscopal effect. Here, we analyzed the antitumor effect of combination therapy using RT with an anti-programmed cell death-1 (PD-1) antibody in primary tumors, using poorly immunogenic metastatic mouse mammary carcinoma 4T1 model. Mice were injected subcutaneously into both flanks with 4T1 cells, and treatment was initiated 12 days later. Mice were randomly assigned to three treatment groups: (1) control (no treatment with RT or immune checkpoint inhibitor (ICI)), (2) RT alone, and (3) RT+ICI. The same RT dose was prescribed in both RT-alone and RT+ICI groups as 10Gy/fx in two fractions and delivered to only one of the two tumor burdens injected at both sides of flanks. In the RT+ICI group, 200 µg fixed dose of PD-1 antibody was intraperitoneally administered concurrently with RT. The RT and ICI combination markedly reduced tumor cell growth not only in the irradiated site but also in non-irradiated sites, a typical characteristic of the abscopal effect. This was observed only in radiation-sensitive cancer cells. Lung metastasis development was lower in RT-irradiated groups (RT-only and RT+ICI groups) than in the non-irradiated group, regardless of the radiation sensitivity of tumor cells. However, there was no additive effect of ICI on RT to control lung metastasis, as was already known regarding the abscopal effect. The combination of local RT with anti-PD-1 blockade could be a promising treatment strategy against metastatic TNBC. Further research is required to integrate our results into a clinical setting.

Comparing syngeneic and autochthonous models of breast cancer to identify tumor immune components that correlate with response to immunotherapy in breast cancer.

BACKGROUND: The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic. METHODS: Three versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model. RESULTS: The 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not. CONCLUSIONS: These studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Dinitrophenol-Hyaluronan Conjugates as Multivalent Antibody-Recruiting Glycopolymers for Targeted Cancer Immunotherapy.

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG3 -DNP]8 , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG3 -DNP]8 significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

Deep Penetration of Nanolevel Drugs and Micrometer-Level T Cells Promoted by Nanomotors for Cancer Immunochemotherapy.

The ability of nanomotors to promote the deep penetration of themselves and the loaded drugs in diseased tissues has been proposed and confirmed. However, whether such motion behavior of the nanomotors can also promote deep penetration of micrometer-sized immune cells in the diseased microenvironment, which is important for the immunotherapy of some diseases, has not been mentioned. Herein, we construct a nitric oxide (NO)-driven nanomotor that can move in the tumor microenvironment, focusing on its motion behavior and the role of NO, the beneficial product released during movement from this kind of nanomotor, in regulating the infiltration behavior and activity of immune cells. It can be found that the drug-loaded nanomotors with both NO-releasing ability and motility can promote the normalization of the tumor vasculature system and the degradation of the intrinsic extracellular matrix (ECM), which can significantly improve the tumor infiltration ability of T cells in vivo. The efficiency of T-cell infiltration in tumor tissue in vivo increased from 2.1 to 28.2%. Both subcutaneous and intraperitoneal implantation tumor models can validate the excellent antitumor effect of drug-loaded NO-driven nanomotors. This combination of motility of the power source from nanomotors and their physiological function offers a design idea for therapeutic agents for the future immunotherapy of many diseases.

Generation of triacyl lipopeptide-modified glycoproteins by metabolic glycoengineering as the neoantigen to boost anti-tumor immune response.

The lack of tumor specific antigens (TSA) and the immune tolerance are two major obstacles for the immunotherapy of cancer. Current immune checkpoint inhibitors (ICIs) show clinical responses in only limited subsets of cancer patients, which, to some extent, depends on the mutation load of tumor cells that may generate neoantigens. Here, we aimed to generate a neoantigen MDP to exhibit stronger anti-tumor efficacy. Methods: In this study, we utilized chemically modified sialic acid precursor tetra acetyl-N-azidoacetyl-mannosamine (AC4ManNAZ) to engineer the glycoproteins on the membranes of tumor cells for the covalent ligation of hapten adjuvant Pam3CSK4 in vivo, which eventually generated a neoantigen, i.e., ManNAZ-DBCO-Pam3CSK4 (MDP), on tumor cells. The high labeling efficiency, relatively specific biodistribution in tumor tissues and the anti-tumor efficacy were confirmed in the syngeneic murine models of the breast cancer and the lung cancer. Results: The generation of MDP neoantigen in tumor-bearing mice significantly evoked both the humoral and the T-cell-dependent antitumor immune responses, resulting in a strong inhibition on the growth of the breast cancer and the lung cancer allografts and significantly prolonged survival of tumor-bearing mice. Interestingly, MDP neoantigen was able to dramatically increase the sensitivity of cancer cells to ICIs and greatly enhance the anti-tumor efficacy in the murine models of both breast cancer and the lung cancer, which showed no or low responses to the immunotherapy with anti-PD1 antibody alone. Conclusions: We developed a simple metabolic glycoengineering method to artificially generate neoantigens on tumor cells to enhance tumor cell immunogenicity, which is able to significantly improve the response and the clinical outcome of ICIs.

Cytotoxic T cells response with decreased CD4/CD8 ratio during mammary tumors inhibition in rats induced by non-contact electric fields.

Background: Breast cancer is the most common cancer in women worldwide and is the leading cause of death in women with cancer. One novel therapy used for breast cancer treatment is non-contact electric fields called electro-capacitive cancer therapy (ECCT) with intermediate frequency (100 kHz) and low intensity (18 Vpp). The objective of this study was to examine the effect of ECCT on mammary tumors growth in rats and observing the immune responses that play a role in fighting the tumor. Methods: Female SD rats were used and divided into four groups, namely control (NINT), placebo (NIT), non- therapy (INT), and therapy (IT) groups with 6 biological replicates in each group. Rats in INT and IT groups were treated with 7,12-dimethylbenz[a]anthracene for mammary tumor induction. Only rats in NIT and IT groups were exposed to ECCT individually for 10 hours per day for 21 days. The size of all tumors was measured with a digital caliper. The distributions of PCNA, ErbB2, caspase-3, CD68, CD4 and CD8-positive cells were observed with immunohistochemistry and scoring with ImageJ. Results: The growth rate of mammary tumors in IT group was significantly lower (p<0.05) than that in the INT group. The number of mitotic figures and the percentage of PCNA, caspase-3, and CD68- positive cells in IT group were significantly lower (p<0.05) than those in INT group. Conversely, the percentage of CD8-positive T cells in IT group was significantly higher (p<0.05) than that in INT group. Moreover, the CD4/CD8 ratio in IT group was decreased. Some tumor tissues were blackened and detached from the surrounding tissue, resulting in an open wound which then healed up upon exposure. Conclusions: Non-contact electric fields exposure showed inhibition on mammary tumor growth in rats while inducing CD8+ T cells that lead to tumor cells death and potentially helps wound healing.

A zwitterionic polypeptide nanocomposite with unique NIR-I/II photoacoustic imaging for NIR-I/II cancer photothermal therapy.

The second near infrared photoacoustic imaging (NIR-II PAI) and photothermal therapy (NIR-II PTT) have attracted wide interest in cancer theranostics because of maximum permission exposure (MPE), deep penetration, and lower scattering and background noise compared to NIR-I counterparts; however, it is imperative to develop biocompatible nanomaterials having NIR-II response. By utilizing multivalent Au-S coordination bonds, we constructed a zwitterionic polypeptide nanocomposite of PMC@AuNP with a suitable size of 48 ± 2 nm, which possessed a strong and broad absorbance at 650-1100 nm and an excellent photothermal conversion efficiency of 49.5%. In vitro biological studies demonstrated that NIR-II PTT within MPE was more effective than NIR-I PTT beyond MPE. Along with X-ray computed tomography and photothermal imaging functions, PMC@AuNP in vivo presented unique NIR-I/II PAI with 2.6-5.9 times signal enhancement compared to the contrast. By single dose and NIR-II irradiation (1064 nm, 1 W cm-2, 10 min), NIR-II PTT within MPE completely eradicated MCF-7 tumors without tissue damage and tumor recurrence within 24 days, inducing a better antitumor efficacy than NIR-I PTT beyond MPE. Importantly, this study provides an innovative method for the fabrication of biocompatible zwitterionic polypeptide nanocomposites with unique NIR-I/II PAI and NIR-II PTT attributes, thus holding great potential for precise cancer theranostics and further clinical transitions.

Systemic metastasis-targeted nanotherapeutic reinforces tumor surgical resection and chemotherapy.

Failure of conventional clinical therapies such as tumor resection and chemotherapy are mainly due to the ineffective control of tumor metastasis. Metastasis consists of three steps: (i) tumor cells extravasate from the primary sites into the circulation system via epithelial-mesenchymal transition (EMT), (ii) the circulating tumor cells (CTCs) form "micro-thrombi" with platelets to evade the immune surveillance in circulation, and (iii) the CTCs colonize in the pre-metastatic niche. Here, we design a systemic metastasis-targeted nanotherapeutic (H@CaPP) composed of an anti-inflammatory agent, piceatannol, and an anti-thrombotic agent, low molecular weight heparin, to hinder the multiple steps of tumor metastasis. H@CaPP is found efficiently impeded EMT, inhibited the formation of "micro-thrombi", and prevented the development of pre-metastatic niche. When combined with surgical resection or chemotherapy, H@CaPP efficiently inhibits tumor metastasis and prolonged overall survival of tumor-bearing mice. Collectively, we provide a simple and effective systemic metastasis-targeted nanotherapeutic for combating tumor metastasis.

Portulaca oleracea L. polysaccharides enhance the immune efficacy of dendritic cell vaccine for breast cancer.

Previous studies have reported that Portulaca oleracea L. polysaccharides (POL-P3b) is an immunoregulatory agent. However, few studies exist on POL-P3b as a novel immune adjuvant in combination with the DC vaccine for breast cancer treatment. In this work, a DC vaccine loaded with mouse 4T1 tumor cell antigen was prepared to evaluate the properties of POL-P3b in inducing the maturation and function of DC derived from mouse bone marrow, and then to investigate the effect of the DC vaccine combined with POL-P3b on breast cancer in vivo and in vitro. Morphological changes of DC were observed using scanning electron microscopy. Phenotypic and functional analyses of DC were detected by flow cytometry and allogeneic lymphocyte reaction. Cytokine levels in the DC culture supernatant were detected by ELISA. Western blotting analysis was used for the protein expression of TLR4, MyD88 and NF-κB. Apoptosis detection and protein expression of the tumor tissue were analyzed by TUNEL staining and immunohistochemistry, respectively. The security of POL-P3b was evaluated by the detection of hematological and blood biochemical indicators and pathological analysis for tissues. POL-P3b can induce DC activation and maturation, which is attributed to increasing the specific anti-tumor immune response, and the mechanism of action involved in the TLR4/MyD88/NF-κB signaling pathway. Experimental results in vivo further suggested that the administration of POL-P3b-treated antigen-primed DC achieved remarkable tumor growth inhibition through inducing apoptosis and enhancing immune responses. Moreover, the POL-P3b-treated DC vaccine was able to inhibit lung metastases. The results proved the feasibility of POL-P3b as an edible adjuvant of the DC vaccine for anti-breast cancer therapy.

Hyperthermia by near infrared radiation induced immune cells activation and infiltration in breast tumor.

Breast cancer is the most common cancer that causes death in women. Conventional therapies, including surgery and chemotherapy, have different therapeutic effects and are commonly associated with risks and side effects. Near infrared radiation is a technique with few side effects that is used for local hyperthermia, typically as an adjuvant to other cancer therapies. The understanding of the use of near NIR as a monotherapy, and its effects on the immune cells activation and infiltration, are limited. In this study, we investigate the effects of HT treatment using NIR on tumor regression and on the immune cells and molecules in breast tumors. Results from this study demonstrated that local HT by NIR at 43 °C reduced tumor progression and significantly increased the median survival of tumor-bearing mice. Immunohistochemical analysis revealed a significant reduction in cells proliferation in treated tumor, which was accompanied by an abundance of heat shock protein 70 (Hsp70). Increased numbers of activated dendritic cells were observed in the draining lymph nodes of the mice, along with infiltration of T cells, NK cells and B cells into the tumor. In contrast, tumor-infiltrated regulatory T cells were largely diminished from the tumor. In addition, higher IFN-γ and IL-2 secretion was observed in tumor of treated mice. Overall, results from this present study extends the understanding of using local HT by NIR to stimulate a favourable immune response against breast cancer.

Semiconducting polymer nano-PROTACs for activatable photo-immunometabolic cancer therapy.

Immunometabolic intervention has been applied to treat cancer via inhibition of certain enzymes associated with intratumoral metabolism. However, small-molecule inhibitors and genetic modification often suffer from insufficiency and off-target side effects. Proteolysis targeting chimeras (PROTACs) provide an alternative way to modulate protein homeostasis for cancer therapy; however, the always-on bioactivity of existing PROTACs potentially leads to uncontrollable protein degradation at non-target sites, limiting their in vivo therapeutic efficacy. We herein report a semiconducting polymer nano-PROTAC (SPNpro) with phototherapeutic and activatable protein degradation abilities for photo-immunometabolic cancer therapy. SPNpro can remotely generate singlet oxygen (1O2) under NIR photoirradiation to eradicate tumor cells and induce immunogenic cell death (ICD) to enhance tumor immunogenicity. Moreover, the PROTAC function of SPNpro is specifically activated by a cancer biomarker (cathepsin B) to trigger targeted proteolysis of immunosuppressive indoleamine 2,3-dioxygenase (IDO) in the tumor of living mice. The persistent IDO degradation blocks tryptophan (Trp)-catabolism program and promotes the activation of effector T cells. Such a SPNpro-mediated in-situ immunometabolic intervention synergizes immunogenic phototherapy to boost the antitumor T-cell immunity, effectively inhibiting tumor growth and metastasis. Thus, this study provides a polymer platform to advance PROTAC in cancer therapy.

Calcium phosphate engineered photosynthetic microalgae to combat hypoxic-tumor by in-situ modulating hypoxia and cascade radio-phototherapy.

Rationale: Hypoxia is one of the crucial restrictions in cancer radiotherapy (RT), which leads to the hypoxia-associated radioresistance of tumor cells and may result in the sharp decline in therapeutic efficacy. Methods: Herein, living photosynthetic microalgae (Chlorella vulgaris, C. vulgaris), were used as oxygenators, for in situ oxygen generation to relieve tumor hypoxia. We engineered the surface of C. vulgaris (CV) cells with calcium phosphate (CaP) shell by biomineralization, to form a biomimetic system (CV@CaP) for efficient tumor delivery and in-situ active photosynthetic oxygenation reaction in tumor. Results: After intravenous injection into tumor-bearing mice, CV@CaP could remarkably alleviate tumor hypoxia by continuous oxygen generation, thereby achieving enhanced radiotherapeutic effect. Furthermore, a cascade phototherapy could be fulfilled by the chlorophyll released from photosynthetic microalgae combined thermal effects under 650 nm laser irradiation. The feasibility of CV@CaP-mediated combinational treatment was finally validated in an orthotropic breast cancer mouse model, revealing its prominent anti-tumor and anti-metastasis efficacy in hypoxic-tumor management. More importantly, the engineered photosynthetic microalgae exhibited excellent fluorescence and photoacoustic imaging properties, allowing the self-monitoring of tumor therapy and tumor microenvironment. Conclusions: Our studies of this photosynthetic microsystem open up a new dimension for solving the radioresistance issue of hypoxic tumors.

Combining losartan with radiotherapy increases tumor control and inhibits lung metastases from a HER2/neu-positive orthotopic breast cancer model.

BACKGROUND: Patients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model. METHODS: We established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm3), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10-14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia. RESULTS: Treatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8-16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation (P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303). In addition, losartan treatment significantly increased tumor vascularity (P = 0.0314) and decreased pimonidazole positive (hypoxic) area (P = 0.0002). CONCLUSIONS: Combining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.

Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.