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Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Gene augmentation therapy entails replacement of the abnormal tumor suppressor genes in cancer cells. In this study, we performed gene augmentation for BRCA1/2 tumor suppressors in order to retard tumor development in breast cancer mouse model. We formulated inorganic carbonate apatite (CA) nanoparticles (NPs) to carry and deliver the purified BRCA1/2 gene- bearing plasmid DNA both in vitro and in vivo. The outcome of BRCA1/2 plasmid-loaded NPs delivery on cellular viability of three breast cancer cell lines such as MCF-7, MDA-MB-231 and 4T1 were evaluated by MTT assay. The result in MCF-7 cell line exhibited that transfection of BRCA 1/2 plasmids with CA NPs significantly reduced cancer cell growth in comparison to control group. Moreover, we noticed a likely pattern of cellular cytotoxicity in 4T1 murine cancer cell line. Following transfection with BRCA1 plasmid-loaded NPs, and Western blot analysis, a notable reduction in the phospho-MAPK protein of MAPK signaling pathway was detected, revealing reduced growth signal. Furthermore, in vivo study in 4T1 induced breast cancer mouse model showed that the tumor growth rate and final volume were decreased significantly in the mouse group treated intravenously with BRCA1 + NPs and BRCA2 + NPs formulations. Our results established that BRCA1/2 plasmids incorporated into CA NPs mitigated breast tumor growth, signifying their application in the therapy for breast cancer., (© 2023. The Author(s).)

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., (© 2021. The Author(s).)

Natural killer (NK) cells are key immune cells engaged in fighting infection and malignant transformation. In this study, we found that canine NK cell-derived exosomes (NK-exosomes) separated from activated cytotoxic NK cell supernatants express specific markers including CD63, CD81, Alix, HSP70, TSG101, Perforin 1, and Granzyme B. We examined the antitumor effects of NK-exosomes in an experimental murine mammary tumor model using REM134 canine mammary carcinoma cell line. We observed changes in tumor size, tumor initiation, progression, and recurrence-related markers in the control, tumor group, and NK-exosome-treated tumor group. We found that the tumor size in the NK-exosome-treated tumor group decreased compared with that of the tumor group in the REM134-driven tumorigenic mouse model. We observed significant changes including the expression of tumorigenesis-related markers, such as B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1), vascular endothelial growth factor (VEGF), matrix metallopeptidase-3 (MMP-3), interleukin-1 β (IL-1 β ), IL-6, tumor necrosis factor- α (TNF- α ), multidrug resistance protein (MDR), tumor suppressor protein p53 (p53), proliferating cell nuclear antigen (PCNA), and the apoptotic markers, B cell lymphoma-2 associated X (Bax) and B cell lymphoma-extra large (Bcl-xL) belonging to the Bcl-2 family, in the tumor group compared with those in the control group. The expression of CD133, a potent cancer stem cell marker, was significantly higher than that of the control. By contrast, the NK-exosome-treated tumor group exhibited a significant reduction in Bmi-1, MMP-3, IL-1 β , IL-6, TNF- α , Bax, Bcl-xL, and PCNA expression compared with that in the tumor group. Furthermore, the expression of CD133, which mediates tumorigenesis, was significantly decreased in the NK-exosome-treated tumor group compared with that in the tumor group. These findings indicate that canine NK-exosomes represent a promising therapeutic tool against canine solid tumors, including mammary carcinoma., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Jienny Lee et al.)

Feline mammary carcinoma (FMC) shows great similarities to human breast cancer in the cellular and molecular levels. So, in cats as in humans, the role of immune responses is indicated to detect and follow up the development of tumors. As a new breast cancer therapeutic approach, Plasmonic Photothermal Therapy (PPTT) is an effective localized treatment for canine and feline mammary-carcinoma. Its systemic effect has not been inquired yet and needs many studies to hypothesis how the PPTT eradicates tumor cells. In this study, it is the first time to detect (P53, PCNA, MUC-1, and C-MYC) feline autoantibodies (AAbs), study the relationship between PCNA AAbs and mammary-tumors, and investigate the effect of PPTT on the humoral immune response of cats with mammary-carcinoma through detection of AAbs level before, during, and after the treatment. The four-AAbs panel was evaluated in serum of normal and clinically diagnosed cats with mammary tumors using Enzyme-Linked Immunosorbent Assay. The panel showed 100% specificity and 93.7% sensitivity to mammary tumors. The panel was evaluated in PPTT monotherapy, mastectomy monotherapy, and combination therapy. PPTT monotherapy decreased AAbs level significantly while mastectomy monotherapy and combination therapy had a nonsignificant effect on AAbs level.

Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) delay progression of metastatic breast cancer. However, complete responses are uncommon and tumors eventually relapse. Here, we show that CDK4/6i can enhance efficacy of T cell-based therapies, such as adoptive T cell transfer or T cell-activating antibodies anti-OX40/anti-4-1BB, in murine breast cancer models. This effect is driven by the induction of chemokines CCL5, CXCL9, and CXCL10 in CDK4/6i-treated tumor cells facilitating recruitment of activated CD8 + T cells, but not Tregs, into the tumor. Mechanistically, chemokine induction is associated with metabolic stress that CDK4/6i treatment induces in breast cancer cells. Despite the cell cycle arrest, CDK4/6i-treated cells retain high metabolic activity driven by deregulated PI3K/mTOR pathway. This causes cell hypertrophy and increases mitochondrial content/activity associated with oxidative stress and inflammatory stress response. Our findings uncover a link between tumor metabolic vulnerabilities and anti-tumor immunity and support further development of CDK4/6i and immunotherapy combinations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: Immunologically quiescent of breast cancer cells has been recognized as the key impediment for the breast cancer immunotherapy. In this study, we aimed to investigate the role of nanoparticle-mediated sonodynamic therapy (SDT) in promoting anti-tumor immune of breast cancer cells and its potential immune mechanisms., Materials and Methods: The phase-transformation nanoparticles (LIP-PFH nanoparticles) were in-house prepared and its physiochemical characters were detected. The CCK-8 assay, apoptosis analysis and Balb/c tumor model establishment were used to explore the anti-tumor effect of LIP-PFH nanoparticles triggered by low-intensity focused ultrasound (LIFU) both in vitro and in vivo. Flow cytometry and immunohistochemistry of CD4 + T, CD8 + T, CD8 + PD-1 + T in blood, spleen and tumor tissue were performed to represent the change of immune response. Detection of immunogenic cell death (ICD) markers was examined to study the potential mechanisms., Results: LIP-PFH nanoparticles triggered by LIFU could inhibit the proliferation and promote the apoptosis of 4T1 cells both in vitro and in vivo. CD4 + T and CD8 + T cell subsets were significantly increased in blood, spleen and tumor tissue, meanwhile CD8 + PD-1 + T cells were reduced, indicating enhancement of anti-tumor immune response of breast cancer cells in the nanoparticle-mediated SDT group. Detection of ICD markers (ATP, high-mobility group box B1, and calreticulin) and flow cytometric analysis of dendritic cell (DC) maturity further showed that the nanoparticle-mediated SDT can promote DC maturation to increase the proportion of cytotoxic T cells by inducing ICD of breast cancer cells., Conclusion: The therapy of nanoparticles-mediated SDT can effectively enhance anti-tumor immune response of breast cancer., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Si et al.)

Gold nanoparticles (AuNPs) are foreseen as a promising tool in nanomedicine, both as drug carriers and radiosensitizers. They have been also proposed as a potential anticancer drug due to the anti-angiogenic effect in tumor tissue. In this work we investigated the effect of citrate-coated AuNPs of nominal diameter 20 nm on the growth and metastatic potential of 4T1 cells originated from a mouse mammary gland tumor inoculated into the mammary fat pad of Balb/ccmdb mice. To evaluate whether AuNPs can prevent the tumor growth, one group of inoculated mice was intragastrically (i.g.) administered with 1 mg/kg of AuNPs daily from day 1 to day 14 after cancer cell implantation. To evaluate whether AuNPs can attenuate the tumor growth, the second group was intravenously (i.v.) administered with 1 or 5 mg/kg of AuNPs, twice on day 5 and day 14 after inoculation. We did not observe any anticancer activity of i.v. nor i.g. administered AuNPs, as they did not affect neither the primary tumor growth rate nor the number of lung metastases. Unexpectedly, both AuNP treatment regimens caused a marked vasodilating effect in the tumor tissue. As no change of potential angiogenic genes ( Fgf2 , Vegfa ) nor inducible nitric oxygenase ( Nos2 ) was observed, we proposed that the vasodilation was caused by AuNP-dependent decomposition of nitrosothiols and direct release of nitric oxide in the tumor tissue.

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Alamsyah F et al.)

Background: Breast cancer is the most common cancer in women worldwide and is the leading cause of death amongst women with cancer. One novel therapy used for breast cancer treatment constitutes non-contact electric fields and is called electro-capacitive cancer therapy (ECCT) with intermediate frequency and low intensity. 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 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 found to have decreased. Some tumor tissues were blackened and detached from the surrounding tissue, resulting in an open wound which then healed upon exposure. Conclusions: Non-contact electric fields exposure showed inhibition on mammary tumor growth in rats while inducing CD8+ T cells, leading to tumor cell death and potentially helping wounds heal., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Alamsyah F et al.)

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication., Competing Interests: No

Breast cancer is the most common malignancy in women. Basic and translational breast cancer research relies heavily on experimental animal models. Ideally, such models for breast cancer should have commonality with human breast cancer in terms of tumor etiology, biological behavior, pathology, and response to therapeutics. This review introduces current progress in different breast cancer experimental animal models and analyzes their characteristics, advantages, disadvantages, and potential applications. Finally, we propose future research directions for breast cancer animal models.

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo , limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal., (©2020 American Association for Cancer Research.)

Currently, there is a huge demand to develop chemoimmunotherapy with reduced systemic toxicity and potent efficacy to combat late-stage cancers with spreading metastases. Here, we report several "cocktail" therapeutic formulations by mixing immunogenic cell death (ICD)-inducing chemotherapeutics and immune adjuvants together with alginate (ALG) for localized chemoimmunotherapy. Immune checkpoint blockade (ICB) antibody may be either included into this cocktail for local injection or used via conventional intravenous injection. After injection of such cocktail into a solid tumor, in-situ gelation of ALG would lead to local retention and sustained release of therapeutics to reduce systemic toxicity. The chemotherapy-induced ICD with the help of immune adjuvant would trigger tumor-specific immune responses, which are further amplified by ICB to elicit potent systemic antitumor immune responses in destructing local tumors, eliminating metastases and inhibiting cancer recurrence. Our strategy of combining clinically used agents for tumor-localized cocktail chemoimmunotherapy possesses great potential for clinical translation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Population-wide testing for cancer-associated mutations has established that more than one-fifth of ovarian and breast carcinomas are associated with inherited risk. Salpingo-oophorectomy and/or mastectomy are currently the only effective options offered to women with high-risk germline mutations. Our goal here is to develop a long-lasting approach that provides immunoprophylaxis for mutation carriers. Our approach leverages the fact that at early stages, tumors recruit hematopoietic stem/progenitor cells (HSPC) from the bone marrow and differentiate them into tumor-supporting cells. We developed a technically simple technology to genetically modify HSPCs in vivo . The technology involves HSPC mobilization and intravenous injection of an integrating HDAd5/35++ vector. In vivo HSPC transduction with a GFP-expressing vector and subsequent implantation of syngeneic tumor cells showed >80% GFP marking in tumor-infiltrating leukocytes. To control expression of transgenes, we developed a miRNA regulation system that is activated only when HSPCs are recruited to and differentiated by the tumor. We tested our approach using the immune checkpoint inhibitor anti-PD-L1-γ1 as an effector gene. In in vivo HSPC-transduced mice with implanted mouse mammary carcinoma (MMC) tumors, after initial tumor growth, tumors regressed and did not recur. Conventional treatment with an anti-PD-L1 mAb had no significant antitumor effect, indicating that early, self-activating expression of anti-PD-L1-γ1 can overcome the immunosuppressive environment in MMC tumors. The efficacy and safety of this approach was further validated in an ovarian cancer model with typical germline mutations (ID8 p53 -/- brca2 -/- ), both in a prophylactic and therapeutic setting. This HSPC gene therapy approach has potential for clinical translation. SIGNIFICANCE: Considering the limited prophylactic options that are currently offered to women with high-risk germ-line mutations, the in vivo HSPC gene therapy approach is a promising strategy that addresses a major medical problem., (©2019 American Association for Cancer Research.)

Residual cell-intrinsic innate immunity in cancer cells hampers infection with oncolytic viruses. Translational control of mRNA is an important feature of innate immunity, yet the identity of translationally regulated mRNAs functioning in host defense remains ill-defined. We report the translatomes of resistant murine "4T1" breast cancer cells infected with three of the most clinically advanced oncolytic viruses: herpes simplex virus 1, reovirus, and vaccinia virus. Common among all three infections are translationally de-repressed mRNAs, including Inpp5e, encoding an inositol 5-phosphatase that modifies lipid second messenger signaling. We find that viral infection induces the expression of an Inpp5e mRNA variant that lacks repressive upstream open reading frames (uORFs) within its 5' leader and is efficiently translated. Furthermore, we show that INPP5E contributes to antiviral immunity by altering virus attachment. These findings uncover a role for translational control through alternative 5' leader expression and assign an antiviral function to the ciliopathy gene Inpp5e., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study identifies mechanisms mediating responses to immune checkpoint inhibitors using mouse models of triple-negative breast cancer. By creating new mammary tumor models, we find that tumor mutation burden and specific immune cells are associated with response. Further, we developed a rich resource of single-cell RNA-seq and bulk mRNA-seq data of immunotherapy-treated and non-treated tumors from sensitive and resistant murine models. Using this, we uncover that immune checkpoint therapy induces T follicular helper cell activation of B cells to facilitate the anti-tumor response in these models. We also show that B cell activation of T cells and the generation of antibody are key to immunotherapy response and propose a new biomarker for immune checkpoint therapy. In total, this work presents resources of new preclinical models of breast cancer with large mRNA-seq and single-cell RNA-seq datasets annotated for sensitivity to therapy and uncovers new components of response to immune checkpoint inhibitors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rationale : Cytotoxic T-lymphocyte-associated antigen 4 (CTLA - 4) is a co-inhibitory checkpoint receptor that is expressed by naïve T-cells in lymph nodes (LNs) to inhibit activation against "self" antigens (Ags). In cancer, anti-CTLA-4 blocks inhibitory action, enabling robust activation of T-cells against tumor Ags presented in tumor draining LNs (TDLNs) . However, anti-CTLA-4 is administered intravenously with limited exposure within TDLNs and immune related adverse events (irAEs) are associated with over-stimulation of the immune system. Methods : Herein, we first deliver anti-CTLA-4 in an orthotopic mammary carcinoma murine model using a nanotopographical microneedle-array device to compare its anti-tumor response to that from systemic administration. Additionally, to demonstrate the feasibility of lymphatic delivery in humans using the device, we use near-infrared fluorescence imaging to image delivery of ICG to LNs. Results : Our data show that lymphatic infusion results in more effective tumor growth inhibition, arrest of metastases, increased tumor infiltrating lymphocytes and complete responses when compared to conventional systemic administration. In clinical studies, we demonstrate for the first time that nanotopographic infusion can deliver ICG through the lymphatics directly to the axilla and inguinal LNs of healthy human volunteers. Conclusion : Taken together, these results suggest that regional delivery using a nanotopography-based microneedle array could revolutionize checkpoint blockade immunotherapy by reducing systemic drug exposure and maximizing drug delivery to TDLNs where tumor Ags present. Future work is needed to determine whether lymphatic delivery of anti-CTLA-4 can alleviate irAEs that occur with systemic dosing., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Earlier, naturally arising mammary cancer in BLRB female mice was shown to reproduce some key pathological characteristics of the familial set of human breast cancer. Then we advanced a novel 3S-paradigm of anticancer research that helped to develop selection criteria and to estimate benefit/risk of local interleukin-2 (IL-2) effects in this spontaneous mouse model. In this paper, the efficacy of single and triple local IL-2 doses is compared using properly selected murine BLRB females based on our previously published data. Only BLRB females bearing spontaneous mammary tumors without subclinical period were used. The tumor growth rate and recipient survival of single and triple IL-2 applications were compared with corresponding parameter values of untreated control. Tumor growth rate was decreased in both experimental groups versus control parameter values. Single IL-2 application resulted in a significant prolongation of the average survival time while triple application caused acute tumor rejection in some females decreasing the survival time of the rest of the recipients. As a result, proper treatment protocol in accurately selected females allowed increasing the complete response rate to 14% in spontaneous mouse model of breast cancer. In conclusion, our approaches may demonstrate the principle methodology developing preselection procedure for breast cancer patients for local IL-2 therapy application.

Background: There are phase 3 clinical trials underway evaluating anti-PD-L1 antibodies as adjuvant (postoperative) monotherapies for resectable renal cell carcinoma (RCC) and triple-negative breast cancer (TNBC); in combination with antiangiogenic VEGF/VEGFR2 inhibitors (e.g., bevacizumab and sunitinib) for metastatic RCC; and in combination with chemotherapeutics as neoadjuvant (preoperative) therapies for resectable TNBC., Methods: This study investigated these and similar clinically relevant drug combinations in highly translational preclinical models of micro- and macro-metastatic disease that spontaneously develop after surgical resection of primary kidney or breast tumours derived from orthotopic implantation of murine cancer cell lines (RENCA luc or EMT-6/CDDP, respectively)., Results: In the RENCA luc model, adjuvant sunitinib plus anti-PD-L1 improved overall survival compared to either drug alone, while the same combination was ineffective as early therapy for unresected primary tumours or late-stage therapy for advanced metastatic disease. In the EMT-6/CDDP model, anti-PD-L1 was highly effective as an adjuvant monotherapy, while its combination with paclitaxel chemotherapy (with or without anti-VEGF) was most effective as a neoadjuvant therapy., Conclusions: Our preclinical data suggest that anti-PD-L1 plus sunitinib may warrant further investigation as an adjuvant therapy for RCC, while anti-PD-L1 may be improved by combining with chemotherapy in the neoadjuvant but not the adjuvant setting of treating breast cancer.

Hypoxia-induced radioresistance is the primary reason for failure of tumor radiotherapy (RT). Changes within the irradiated tumor microenvironment (TME) including oxygen, reactive oxygen species (ROS) and nitric oxide (NO) are closely related to radioresistance. Therefore, there is an urgent need to develop new approaches for overcoming hypoxic radioresistance by incorporating TME regulation into current radiotherapeutic strategies., Methods: Herein, we explored a radiation-responsive nanotheranostic system to enhance RT effects on hypoxic tumors by multi-way therapeutic effects. This system was developed by loading S-nitrosothiol groups (SNO, a NO donor) and indocyanine green (ICG, a photosensitizer) onto mesoporous silica shells of Eu 3+ -doped NaGdF 4 scintillating nanocrystals (NSC)., Results: Under X-ray radiation, this system can increase the local dosage by high-Z elements, promote ROS generation by X-ray-induced photodynamic therapy, and produce high levels of NO to enhance tumor-killing effects and improve hypoxia via NO-induced vasodilation. In vitro and in vivo studies revealed that this combined strategy can greatly reinforce DNA damage and apoptosis of hypoxic tumor cells, while significantly suppressing tumor growth, improving tumor hypoxia and promoting p53 up-regulation and HIF1α down-regulation. In addition, this system showed pronounced tumor contrast performance in T 1 -weighted magnetic resonance imaging and computed tomography., Conclusion: This work demonstrates the great potential of scintillating nanotheranostics for multimodal imaging-guided X-ray radiation-triggered tumor combined therapy to overcome radioresistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: In order to explore the possibility of treating breast cancer by local photo-therapy, a photothermal agents loaded in situ hydrogel was established. In detail, The Cu 2 MnS 2 nanoplates were prepared by one-pot synthesis and, the thermosensitive Pluronic F127 was used as the hydrogel matrix. The Cu 2 MnS 2 nanoplates and the hydrogel were characterized by morphous, particle size, serum stability, photothermal performance upon repeated 808 nm laser irradiation as well as the rheology features. The therapeutic effects of the Cu 2 MnS 2 nanoplates and the hydrogel were evaluated qualitatively and quantitatively in 4T1 mouse breast cancer cells. The retention, photothermal efficacy, therapeutic effects and systemic toxicity of the hydrogel were assessed in tumor bearing mouse model., Results: The Cu 2 MnS 2 nanoplates with a diameter of about 35 nm exhibited satisfying serum stability, photo-heat conversion ability and repeated laser exposure stability. The hydrogel encapsulation did not negatively influence the above features of the photothermal agent. The nanoplates loaded in situ hydrogel shows a phase transition at body temperature and, as a result, a long retention in vivo., Conclusions: The photothermal agent embedded hydrogel played a promising photothermal therapeutic effects in tumor bearing mouse model with low systemic toxicity after peritumoral administration.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu + mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3 + tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed. Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately. Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA. Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Metastatic triple-negative breast cancer (mTNBC) is an aggressive disease among women worldwide, characterized by high mortality and poor prognosis despite systemic therapy with radiation and chemotherapies. Photodynamic therapy (PDT) is an important strategy to eliminate the primary tumor, however its therapeutic efficacy against metastases and recurrence is still limited. Here, we employed a template method to develop the core-shell gold nanocage@manganese dioxide (AuNC@MnO 2 , AM) nanoparticles as tumor microenvironment responsive oxygen producers and near-infrared (NIR)-triggered reactive oxygen species (ROS) generators for oxygen-boosted immunogenic PDT against mTNBC. In this platform, MnO 2 shell degrades in acidic tumor microenvironment pH/H 2 O 2 conditions and generates massive oxygen to boost PDT effect of AM nanoparticles under laser irradiation. Fluorescence (FL)/photoacoustic (PA)/magnetic resonance (MR) multimodal imaging confirms the effective accumulation of AM nanoparticles with sufficient oxygenation in tumor site to ameliorate local hypoxia. Moreover, the oxygen-boosted PDT effect of AM not only destroys primary tumor effectively but also elicits immunogenic cell death (ICD) with damage-associated molecular patterns (DAMPs) release, which subsequently induces DC maturation and effector cells activation, thereby robustly evoking systematic antitumor immune responses against mTNBC. Hence, this oxygen-boosted immunogenic PDT nanosystem offers a promising approach to ablate primary tumor and simultaneously prevent tumor metastases via immunogenic abscopal effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nogo-B receptor (NgBR) plays fundamental roles in regulating angiogenesis, vascular development, and the epithelial-mesenchymal transition (EMT) of cancer cells. However, the therapeutic effect of NgBR blockade on tumor vasculature and malignancy is unknown, investigations on which requires an adequate delivery system for small interfering RNA against NgBR (NgBR siRNA). Here a surface charge switchable polymeric nanoparticle that was sensitive to the slightly acidic tumor microenvironment was developed for steady delivery of NgBR siRNA to tumor tissues. The nanoformulation was constructed by conjugating 2, 3-dimethylmaleic anhydride (DMMA) molecules to the surface amines of micelles formed by cationic co-polymer poly(lactic-co-glycolic acid) 2 -poly(ethylenimine) and subsequent absorption of NgBR siRNAs. The nanoparticles remained negatively charged in physiological condition and smartly converted to positive surface charge due to tumor-acidity-activated shedding of DMMA. The charge conversion facilitated cellular uptake of siRNAs and in turn efficiently depleted the expression of NgBR in tumor-bearing tissues. Silencing of NgBR suppressed endothelial cell migration and tubule formation, and reverted the EMT process of breast cancer cells. Delivery of the nanoformulation to mice bearing orthotopic breast carcinoma showed no effect on tumor growth, but led to remarkable decrease of distant metastasis by normalizing tumor vessels and suppressing the EMT of breast cancer cells. This study demonstrated that NgBR is a promising therapeutic target in abnormal tumor vasculature and aggressive cancer cells, and the tumor-responsive nanoparticle with the feature of charge transformation offers great potential for tumor-specific delivery of gene therapeutics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Basal-like breast cancer (BLBC) is a malignant carcinoma with aggressive motility and rapid growth. Accounting for 15% of breast cancers, BLBC often exhibits a poor prognosis and tends to metastasize to the brain and lungs. Because most BLBC display a triple-negative phenotype (ER-, PR-, and HER2-), conventional cytotoxic chemotherapy remains the only treatment option despite poor success and high rate of relapse. The overexpression of the forkhead-box transcription factor C1 (FOXC1) was recently identified as a biomarker of BLBC. Increased expression of FOXC1 was linked to excessive mobility and growth of BLBC cell lines, suggesting FOXC1 as a therapeutic target. In this study, siRNA-mediated knockdown of FOXC1 was confirmed to decrease the proliferation rate, migration, and invasion in a model BLBC-like cell line (4T1). 4T1 and 4T1-∆FOXC1 cells lacking FOXC1 expression (generated by CRISPR/Cas9) were used to evaluate the effects of FOXC1 expression in an orthotopic murine model of BLBC. No statistically significant difference in tumor volume was observed between 4T1 and 4T1-∆FOXC1 tumors. Furthermore, tumors metastasized to the liver and lungs to a similar degree regardless of FOXC1 expression. These data suggest that, despite positive results in vitro, FOXC1 may not be a promising therapeutic target for BLBC.

Background : The emergence of two-dimensional MXenes has spurred their versatile applications in broad fields, but the exploring of novel MXene-based family members and their potential applications in theranostic nanomedicine (concurrent diagnostic imaging and therapy) have been rarely explored. In this work, we report the construction of a novel superparamagnetic MXene-based theranostic nanoplatform for efficient breast-cancer theranostics, which was based on intriguing tantalum carbide (Ta 4 C 3 ) MXene and its further rational surface-superparamagnetic iron-oxide functionalization (Ta 4 C 3 -IONP-SPs composite MXenes) for efficient breast-cancer theranostic. Methods: The fabrication of ultrathin Ta 4 C 3 nanosheets was based on an exfoliation strategy and superparamagnetic iron oxide nanoparticles were in-situ grown onto the surface of Ta 4 C 3 MXene according to the redox reaction of MXene. Ta 4 C 3 -IONP MXenes were modified with soybean phospholipid (SP) to guarantee high stability in physiological conditions. The photothermal therapy, contrast-enhanced CT, T 2 -weighted magnetic resonance imaging and the high biocompatibility of these composite nanosheets have also been evaluated in vitro at cellular level and in vivo on mice breast tumor allograft tumor model. Results: The Ta component of Ta 4 C 3 -IONP-SPs exhibits high performance for contrast-enhanced CT imaging because of its high atomic number and high X-ray attenuation coefficient, and the integrated superparamagnetic IONPs act as excellent contrast agents for T 2 -weighted magnetic resonance imaging. Especially, these Ta 4 C 3 -IONP-SPs composite nanosheets with high photothermal-conversion efficiency ( η : 32.5%) has achieved complete tumor eradication without reoccurrence, verifying their highly efficient breast-tumor photo-ablation performance. Conclusion: This work not only significantly broadens the biomedical applications of MXene-based nanoplatforms (Ta 4 C 3 MXene) by exploring their novel family members and further functionalization strategies (magnetic functionalization in this work), but also provides a novel and efficient theranostic nanoplatform for efficient breast-cancer theranostics., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment., (Copyright © 2017. Published by Elsevier B.V.)

Aim: To evaluate the effect of intraperitoneal injections of heat-killed Lactobacillus crispatus on breast tumor size and overall survival of Balb/c mouse received 4T1 mammary carcinoma. Materials and methods: Different doses of L. crispatus have been injected intraperitoneally in BALB/c mice., Results: Tumor size was decreased in the experiment group treated with 1 × 10 8 bacteria/200 μl. Treatment with 1 × 10 8 bacteria/200 μl resulted in survival improvement. The myeloid-derived suppressor cells and reactive oxygen species production have been increased in all groups. Cox2 expression decreased in tumor tissues of the mice treated with 10 8 bacteria/200 μl. The expressions of Arginase and iNos increased in the spleen and tumor tissues of those treated with 5 × 10 8 bacteria/200 μl., Conclusion: We have shown the protective effect of L. crispatus on survival of tumor-bearing mice.

Near-infrared (NIR) laser-induced photothermal therapy (PTT) uses a photothermal agent to convert optical energy into thermal energy and has great potential as an effective local, minimally invasive treatment modality for killing cancer cells. To improve the efficacy of PTT, we developed poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulating superparamagnetic iron oxide (Fe 3 O 4 ), indocyanine green (ICG), and perfluoropentane (PFP) as synergistic agents for NIR laser-induced PTT. We fabricated a novel type of phase-shifting fluorescent magnetic NPs, Fe 3 O 4 /ICG@PLGA/PFP NPs, that effectively produce heat in response to NIR laser irradiation for an enhanced thermal ablation effect and a phase-shift thermoelastic expansion effect, and thus, can be used as a photothermal agent. After in vitro treatment of MCF-7 breast cancer cells with Fe 3 O 4 /ICG@PLGA/PFP NPs and NIR laser irradiation, histology and electron microscopy confirmed severe damage to the cells and the formation of many microbubbles with iron particles at the edge or outside of the microbubbles. In vivo experiments in mice with MCF-7 tumors demonstrated that Fe 3 O 4 /ICG@PLGA/PFP NPs could achieve tumor ablation upon NIR laser irradiation with minimal toxicity to non-irradiated tissues. Together, our results indicate that Fe 3 O 4 /ICG@PLGA/PFP NPs can be used as effective nanotheranostic agents for tumor ablation.

Purpose: Triple-negative breast cancer (TNBC) represents a cancer stem cell-enriched phenotype. Hypoxia-inducible factor-1α (HIF-1α) induces the expression of proteins associated with stemness and is highly upregulated in TNBC. We questioned whether HIF-1α was immunogenic and whether vaccination targeting HIF-1α would impact the growth of basal-like mammary tumors in transgenic mice. Experimental Design: We evaluated HIF-1α-specific IgG in sera from controls and patients with breast cancer. Class II epitopes derived from the HIF-1α protein sequence were validated by ELISPOT. To assess therapeutic efficacy, we immunized Tg-MMTVneu and C3(1)Tag mice with HIF-1α Th1-inducing peptides. Stem cells were isolated via magnetic bead separation. Levels of HIF-1α and stem cells in the tumor were quantitated by Western blotting and flow cytometry. Results: The magnitude ( P < 0.001) and incidence ( P < 0.001) of HIF-1α-specific IgG were elevated in TNBC patients compared with controls. Both breast cancer patients and donors showed evidence of HIF-1α-specific Th1 and Th2 immunity. Three HIF-1α-specific Th1 class II restricted epitopes that were highly homologous between species elicited type I immunity in mice. After HIF-1α vaccination, mammary tumor growth was significantly inhibited in only C3(1)Tag (basal-like/stem cell high ; P < 0.001) not TgMMTV-neu (luminal/neu/stem cell low ; P = 0.859) murine models. Vaccination increased type I T cells in the tumor ( P = 0.001) and decreased cells expressing the stem cell marker, Sca-1, compared with controls ( P = 0.004). Conclusions: An HIF-1α vaccine may be uniquely effective in limiting tumor growth in TNBC. Inhibiting outgrowth of breast cancer stem cells via active immunization in the adjuvant setting may impact disease recurrence. Clin Cancer Res; 23(13); 3396-404. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Two of the challenges for clinical implementation of nano-therapeutic strategies are optimization of tumor targeting and clearance of the nanoagents in vivo. Herein, a cell-mediated therapy by transporting 2D Bi 2 Se 3 nanosheets within macrophage vehicles is described. The Bi 2 Se 3 nanosheets with excellent near-infrared photothermal performance exhibit high macrophage uptake and negligible cytotoxicity thus facilitating the fabrication of Bi 2 Se 3 -laden-macrophages. Compared with bare Bi 2 Se 3 , the Bi 2 Se 3 -laden-macrophages after intravenous injection show prolonged blood circulation and can overcome the hypoxia-associated drug delivery barrier to target the tumor efficiently and dramatically enhance the efficiency of photothermal cancer therapy. The Bi 2 Se 3 -laden-macrophages possess good biocompatibility as demonstrated by the biochemical and histological analyses and furthermore, most of the materials are excreted from the body within 25 days. Our findings reveal a desirable system for highly efficient near-infrared photothermal cancer therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

RCAS avian viruses have been used to deliver oncogene expression and induce tumors in transgenic mice expressing the virus receptor TVA. Here we report the generation and characterization of a novel RCAS-Cre-IRES-PyMT (RCI-PyMT) virus designed to specifically knockout genes of interest in tumors generated in appropriate mutant mouse hosts. FGF receptor 1 (FGFR1) is a gene that is amplified in human breast cancer, but there have been no definitive studies on its function in mammary tumorigenesis, progression, and metastasis in vivo in spontaneous tumors in mice. We used the retroviral tumor knockout, or TuKO, strategy to delete fgfr1 in PyMT-induced mammary tumors in K19-tva/fgfr1 loxP/loxP mice. The similarly injected control K19-tva mice developed mammary tumors exhibiting high metastasis to lung, making this an ideal model for breast cancer metastasis. The fgfr1 TuKO tumors showed significantly decreased primary tumor growth and, most importantly, greatly reduced metastasis to lung. In contrast to previous reports, FGFR1 action in this spontaneous mammary tumor model does not significantly induce epithelial-to-mesenchymal transition. Loss of FGFR1 does generate a gene signature that is reverse correlated with FGFR1 gene amplification and/or upregulation in human breast cancer. Our results suggest that FGFR1 signaling is a key pathway driving breast cancer lung metastasis and that targeting FGFR1 in breast cancer is an exciting approach to inhibit metastasis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Sonodynamic therapy (SDT) kills tumor cells through the synergistic effects of ultrasound (US) and a sonosensitizer agent. 5-Aminolevulinic acid (5-ALA) has been used as a sonodynamic sensitizer for cancer treatment. However, studies have shown that 5-ALA-based SDT has limited efficacy against malignant tumors. In this study, we examined whether artesunate (ART) could enhance the cytotoxicity of 5-ALA-based SDT against mouse mammary tumor (EMT-6) cells in vitro. In the ART, ART + US, ART + 5-ALA, and ART + 5-ALA + US groups, the cell survival rate correlated with ART concentration, and decreased with increasing concentrations of ART. Morphologically, many apoptotic and necrotic cells were observed in the ART + 5-ALA + US group. The percentage of reactive oxygen species-positive cells in the ART + 5-ALA + US group was also significantly higher than that in the 5-ALA group (p = 0.0228), and the cell death induced by ART + 5-ALA + US could be inhibited by the antioxidant N-acetylcysteine. These results show that ART offers great potential in enhancing the efficacy of 5-ALA-based SDT for the treatment of cancer. However, these results are only based on in vitro studies, and further in vivo studies are required.

Background: Minimally invasive therapies, such as microwave ablation (MWA), are widely used for the treatment of solid tumors. Previous studies suggest that MWA is feasible for the treatment of small breast cancer, and thermal ablation may induce adaptive antitumor immunity. However, the induced immune responses are mostly weak, and the immunomodulation effects of MWA in breast cancer are unclear. Immunostimulant OK-432 can induce tumor-specific T-cell responses and may augment the immunity induced by MWA., Methods: We treated 4T1 breast cancer bearing BALB/c mice with MWA, OK-432, MWA plus OK-432, or left without treatment. Survival time was evaluated with the Kaplan-Meyer method comparing survival curves by log-rank test. On day 25 after ablation, surviving mice received tumor rechallenge, and the rechallenged tumor volumes were calculated every 5 days. Immunohistochemistry and flow cytometry were used to evaluate the T-cell immune responses in ablated tissues and spleens. The tumor-specific immunity was assessed by enzyme-linked immunospot assays. Besides, the cytokine patterns were identified from enzyme-linked immunosorbent assay., Results: Microwave ablation plus OK-432 resulted in longer survival than single treatment and protect most surviving mice from tumor rechallenge. Both local and systemic T-cell responses were induced by MWA and were further enhanced by subsequent administration of OK-432. Moreover, the combination of MWA and OK-432 induced stronger tumor-specific immune responses than MWA alone. In addition, OK-432 and MWA synergistically promoted the production of Th1-type but not Th2-type cytokines, and polarized T-cell responses to Th1-dominant state., Conclusions: The T-cell immune responses were activated by MWA in breast cancer. Furthermore, the combination of MWA and OK-432 induced Th1-type response and elicited specific antitumor immunity.

It is well-known that vaccines comprising of irradiated whole tumor cells or tumor-derived heat shock proteins can generate tumor-specific immune responses. In contrast, we showed recently that vaccines composed of autophagosomes (DRibbles) derived from syngeneic sarcomas could induce cross-reactive T-cell responses and cross-protection against the tumor. This unusual property of DRibbles was related to the selective recruitment of defective ribosomal products (DRiPs) and other short-lived proteins (SLiPs) into autophagosomes via sequestosome (SQSTM1, p62) mediated association of ubiquitinated SLiPs to the autophagy gene product LC3. Here, we extend our observations to mammary carcinomas from mice of different genetic background. We demonstrated that combined of intranodal administration of autologous or allogeneic DRibbles together with anti-OX40 antibody led to robust proliferation, expansion, and differentiation of memory and effector T cells. We also showed that SLiPs is an excellent source of antigen for cross-priming of CD8 + T-cells that recognize shared tumor antigens in the context of host MHC class I molecules. Thus, our results provide a strong basis for novel clinical trials that combine allogeneic "off-the-shelf" DRibble vaccines together with antibodies against co-stimulatory molecules., Competing Interests: Hong-Ming Hu and Bernard A. Fox are co-founders of UbiVac, which has licensed the autophagosome intellectual property. Andrew D. Weinberg and Nicholas P. Morris are founder and employee of AgonOx, which has an ownership interest in OX40 patents. No potential conflicts of interest were disclosed by the other authors.

OBJECTIVE To describe the prevalence, histologic characteristics, concomitant abnormalities, and outcomes for various types of mammary gland tumors in companion rats (Rattus norvegicus). DESIGN Retrospective case series. ANIMALS 100 client-owned rats. PROCEDURES Medical records of companion rats that had an SC mass and were examined at a veterinary teaching hospital between 1990 and 2015 were reviewed. Information regarding the signalment, age at mass detection, reproductive sterilization status, histologic diagnosis of the SC mass, location of the initial and all subsequent SC masses, treatments administered, and clinical outcomes was extracted from each record and summarized. RESULTS 105 SC masses were initially detected in 100 rats. The most prevalent SC mass identified was mammary gland fibroadenoma (56/105 [53%]), followed by mammary gland carcinoma (13/105 [12%]). Overall, 26 of 105 (25%) masses were malignant. Sexually intact males were more likely to have nonmammary SC tumors than sexually intact females. In rats receiving no adjunctive treatment after excision of a mammary gland fibroadenoma (n = 16), a second fibroadenoma was detected 1 to 8 months after initial excision, at a median of 4.5 months after surgery. A concomitant pituitary gland tumor was identified in most rats with mammary gland fibroadenoma (21/28 [75%]) and other types of mammary gland tumors (10/17 [59%]). Fourteen of 35 (40%) rats with mammary gland fibroadenoma had concomitant reproductive tract abnormalities. CONCLUSION AND CLINICAL RELEVANCE Results suggested that, like other species, companion rats with SC masses should undergo a thorough diagnostic workup that includes histologic examination of the excised mass.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Long non-coding RNAs (lncRNAs) represent the largest and most diverse class of non-coding RNAs, comprising almost 16,000 currently annotated transcripts in human and 10,000 in mouse. Here, we investigated the role of lncRNAs in mammary tumors by performing RNA-seq on tumor sections and organoids derived from MMTV-PyMT and MMTV-Neu-NDL mice. We identified several hundred lncRNAs that were overexpressed compared to normal mammary epithelium. Among these potentially oncogenic lncRNAs we prioritized a subset as Mammary Tumor Associated RNAs (MaTARs) and determined their human counterparts, hMaTARs. To functionally validate the role of MaTARs, we performed antisense knockdown and observed reduced cell proliferation, invasion, and/or organoid branching in a cancer-specific context. Assessing the expression of hMaTARs in human breast tumors revealed that 19 hMaTARs are significantly upregulated and many of these correlate with breast cancer subtype and/or hormone receptor status, indicating potential clinical relevance., Competing Interests: D.L.S. receives research support from, and is a consultant to, Ionis Pharmaceuticals., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plasmonic photothermal therapy (PPTT) is a cancer therapy in which gold nanorods are injected at the site of a tumor before near-infrared light is transiently applied to the tumor causing localized cell death. Previously, PPTT studies have been carried out on xenograft mice models. Herein, we report a study showing the feasibility of PPTT as applied to natural tumors in the mammary glands of dogs and cats, which more realistically represent their human equivalents at the molecular level. We optimized a regime of three low PPTT doses at 2-week intervals that ablated tumors mainly via apoptosis in 13 natural mammary gland tumors from seven animals. Histopathology, X-ray, blood profiles, and comprehensive examinations were used for both the diagnosis and the evaluation of tumor statuses before and after treatment. Histopathology results showed an obvious reduction in the cancer grade shortly after the first treatment and a complete regression after the third treatment. Blood tests showed no obvious change in liver and kidney functions. Similarly, X-ray diffraction showed no metastasis after 1 year of treatment. In conclusion, our study suggests the feasibility of applying the gold nanorods-PPTT on natural tumors in dogs and cats without any relapse or toxicity effects after 1 year of treatment.

We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL- 2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemoattraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12- producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor.

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45-, showed a high tumorigenicity compared to non-CD24+CD90+CD45- cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45- TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45- cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45- cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas., Competing Interests: Conflicts of Interest: None declared.

Gene electrotransfer of plasmid encoding shRNA against endoglin exerts antitumor efficacy, predominantly by vascular targeted effect. As vascular targeting therapies can promote radiosensitization, the aim of this study was to explore this gene therapy approach with single and split dose of irradiation in an endoglin non-expressing TS/A mammary adenocarcinoma tumor model to specifically study the vascular effects. Intratumoral gene electrotransfer of plasmids encoding shRNA against endoglin, under the control of a constitutive or tissue-specific promoter for endothelial cells, combined with a single or three split doses of irradiations was evaluated for the antitumor efficacy and histologically. Both plasmids proved to be equally effective in tumor radiosensitization with 40-47% of tumor cures. The combined treatment induced a significant decrease in the number of blood vessels and proliferating cells, and an increase in levels of necrosis, apoptosis and hypoxia; therefore, the antitumor efficacy was ascribed to the interaction of vascular targeted effect of gene therapy with irradiation. Endoglin silencing by the shRNA technology, combined with electrotransfer and the use of a tissue-specific promoter for endothelial cells, proved to be a feasible and effective therapeutic approach that can be used in combined treatment with tumor irradiation.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

Two-dimensional MoS2 nanosheet has been extensively explored as a photothermal agent for tumor regression; however, its surface modification remains a great challenge. Herein, as an alternative to surface polyethylene glycol modification (PEGylation), a facile approach based on "thin-film" strategy has been proposed for the first time to produce soybean phospholipid-encapsulated MoS2 (SP-MoS2) nanosheets. By simply vacuum-treating MoS2 nanosheets/soybean phospholipid/chloroform dispersion in a rotary evaporator, SP-MoS2 nanosheet was successfully constructed. Owing to the steric hindrance of polymer chains, the surface-coated soybean phospholipid endowed MoS2 nanosheets with excellent colloidal stability. Without showing detectable in vitro and in vivo hemolysis, coagulation, and cyto-/histotoxicity, the constructed SP-MoS2 nanosheets showed good photothermal conversion performance and photothermal stability. SP-MoS2 nanosheet was shown to be a promising platform for in vitro and in vivo breast tumor photothermal therapy. The produced SP-MoS2 nanosheets featured low cost, simple fabrication, and good in vivo hemo-/histocompatibility and hold promising potential for future clinical tumor therapy.

Our evolving understandings of cell-material interactions provide insights for using polymers to modulate cell behaviour that may lead to therapeutic applications. It is known that in certain cancers, myeloid-derived suppressor cells (MDSCs) play vital roles in promoting tumour progression, chiefly because of their 'alternatively activated' (or M2) phenotype that orchestrates immunosuppression. In this study, we demonstrated that two cationic polymers - cationic dextran (C-dextran) and polyethyleneimine (PEI) - could directly remodel these cells into an anti-tumour, 'classically activated' (or M1) phenotype, thereby stimulating these cells to express tumouricidal cytokines, reactivating the T cell functions, and prolonging the lifespan of the mice model. Our investigations with knock-out mice further indicate that the functions of these cationic polymers require the involvement of toll-like receptor 4-mediated signalling. Taken together, our study suggests that these cationic polymers can effectively and directly re-polarize MDSCs from an immunosuppressive characteristic to an anti-tumour phenotype, leading to successful restoration of immune surveillance in the tumour microenvironment and elimination of tumour cells. Our findings may have immediate impact on further development of polymer-based therapeutics for cancer immunotherapy.

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti-cytotoxic T-lymphocyte-associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti-DEC-205 (dendritic and epithelial cells, 205 kDa)-HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC50) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.