PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

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.

Canine Natural Killer Cell-Derived Exosomes Exhibit Antitumor Activity in a Mouse Model of Canine Mammary Tumor.

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-1ß (IL-1ß), 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-1ß, 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.

Submicron particle docetaxel intratumoral injection in combination with anti-mCTLA-4 into 4T1-Luc orthotopic implants reduces primary tumor and metastatic pulmonary lesions.

We describe here characterization of the response of local and metastatic disease and immunomodulation following intratumoral (IT) injection of submicron particle docetaxel (SPD) administered alone or in combination with systemic antibody anti-mCTLA-4 (anti-mCTLA-4) in the metastatic 4T1-Luc2-1A4 (4T1) murine breast cancer model. In-life assessments of treatment tolerance, tumor volume (TV), and metastasis were performed (n = 10 animals/group). At study end, immune cell populations in tumor-site tissues and peripheral blood were analyzed using flow cytometry. Signs of distress typical of this aggressive tumor model occurred across all animals except for the combination treated which were asymptomatic and gained weight. TV at study end was significantly reduced in the combination group versus untreated [43% reduced (p < 0.05)] and vehicle controls [54% reduced (p < 0.0001)]. No evidence of thoracic metastasis was found in 40% of combination group animals and thoracic bioluminescence imaging (BLI) was reduced vs. untreated controls (p < 0.01). Significant elevations (p < 0.05) in CD4 + T, CD4 + helper T, Treg, and NKT cells were found in tumor and blood in SPD or combination treatment compared to controls or anti-mCTLA-4. Combination treatment increased tumor-associated CD8 + T cells (p < 0.01), peripheral B cells (p < 0.01), and tumor associated and circulating dendritic cells (DC) (p < 0.05). Tumor-associated NK cells were significantly increased in SPD ± anti-mCTLA-4 treatments (p < 0.01). Myeloid-derived suppressor cells (MDSC) were reduced in bloods in SPD ± anti-mCTLA-4 groups (p < 0.05). These data demonstrate that both SPD and anti-mCTLA-4 produce local anti-tumor effects as well as reductions in metastasis which are significantly enhanced when administered in combination.

Reduced infiltration of regulatory T cells in tumours from mice fed daily with gamma-tocotrienol supplementation.

Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T helper (Th) and T regulatory cell (Treg ) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2 weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were euthanized. Mice (n = 6) were euthanized at specified time-points for various analysis (blood leucocyte, cytokine production and immunohistochemistry). Tumour volume was measured once every 7 days. Gene expression studies were carried out on tumour-specific T lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p < 0.05), CD8+ (p < 0.05) T-cells and natural killer cells (p < 0.05) but suppressed Treg cells (p < 0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon (IFN)-γ and lower transforming growth factor (TGF)-ꞵ levels were noted in the γT3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-12ꞵ2R), interleukin (IL)-24 and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the γT3-fed animals. Gene expression studies showed the down-regulation of seven prominent genes in splenic CD4+ T cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T cell-dependent cell-mediated immune responses and suppressed T cells in the tumour microenvironment in a syngeneic mouse model of breast cancer.

Pathological and Immunohistochemical Microscopy of Natural Cases of Canine and Feline Neoplastic Mammary Lesions.

Mammary cancer is the second most common tumor worldwide. Small animal mammary neoplasms provide an outstanding model to study cancer in humans, as tumors in both share a similar environment, histopathologic features, and biological behavior. This study aims to investigate the percentage and microscopy of breast tumors in affected dogs and cats; its relationship to breed, age, and sex; and the immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), Ki-67, and cytokeratin 8. Twenty-four females (12 dogs and 12 cats) and one male were examined from February 2018 to February 2020. The highest percentage of mammary neoplasia from the highest to the lowest manifested as tubular carcinoma, leiomyosarcoma, fibroadenoma, and cystic papillary carcinoma. The current study reported the second micropapillary invasive carcinoma in a male cat and the third lipid-rich carcinoma in a female cat. Although tubular carcinoma was the most common mammary neoplasm in cats, leiomyosarcoma was the most common in dogs. The immunohistochemical staining revealed diffuse and intense cytoplasmic immunoreactivity for cytokeratin 8 in lipid-rich carcinomas. However, moderate expression of ER in benign tumors and slight to moderate ER expression in malignant mammary lesions were reported. On the contrary, there was a negative PR expression in benign lesion. It could be concluded that a close relationship between ER expression and nuclear antigen Ki-67 was found.

The implication of autoantibodies in early diagnosis and monitoring of plasmonic photothermal therapy in the treatment of feline mammary carcinoma.

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.

Metabolic modulation by CDK4/6 inhibitor promotes chemokine-mediated recruitment of T cells into mammary tumors.

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.

Phase-Transformation Nanoparticle-Mediated Sonodynamic Therapy: An Effective Modality to Enhance Anti-Tumor Immune Response by Inducing Immunogenic Cell Death in Breast Cancer.

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.

M1 and M2 tumour-associated macrophages subsets in canine malignant mammary tumours: An immunohistochemical study.

Among the innate and adaptative immune cells recruited to the tumour site, tumour associated macrophages (TAMs) are particularly abundant and by simplified classification can be classified into (M1) and (M2) TAMs. In the present study, we quantified by immunohistochemistry ionized calcium binding adaptor molecule 1 (Iba1)-positive total and CD204-positive M2-polarized TAMs in 60 canine malignant mammary tumours (CMMTs) to analyze the relationship between M1 or M2 response and the histopathologic features of examined CMMTs, the dogs' body condition score (BCS) and the progression of the neoplastic disease. The mean number of total and CD204+ TAMS were significantly higher in solid and in grade III than in grades I and II carcinomas. Moreover, the mean number of CD204-positive TAMs was significantly higher in CMMTs with lymphatic invasion and necrosis rather than CMMTs without. The presence of higher number of CD204-positive M2-polarized TAMs was associated with a worst outcome of the neoplastic disease: bitches bearing CMMTs with a prevalent M2-polarized TAM response had a median cancer-specific survival time of 449 days, while in animals with a M1-polarized TAM response the median cancer-specific survival time was 1209 days. The results of our study confirm that in CMMTs the presence of a M2-polarized TAMs response might affect the tumour development and behaviour. Finally, it strongly suggests the potential of CD204 expression as a prognostic factor.

Protective immune response against P32 oncogenic peptide-pulsed PBMCs in mouse models of breast cancer.

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

Clinical significance and prognostic role of tumor-associated macrophages infiltration according to histologic location in canine mammary carcinomas.

Tumor-associated macrophages (TAMs) have been involved in growth and metastases of human and canine mammary tumors. However, the prognostic importance of TAM specific location in canine mammary tumors (CMT) was not evaluated. In this study, we evaluated the potential role of TAMs in specific histologic locations - intratumoral (iTAM) and stromal (sTAM), as well as total macrophage (tTAM) counts - as prognostic indicators in CMT. Clinicopathologic data from 66 animals with mammary carcinoma and their tumors were used in this study. Samples were stained with anti-macrophage antibody for subsequent TAM count. High levels of iTAM, sTAM, and tTAM were related with advanced clinical stage and vascular invasion. Additionally, tTAM revealed a relation with larger tumor size, while high levels of sTAM and tTAM were also correlated with node metastasis and a poor prognosis based on survival analysis. CMT with aggressive features can reveal higher TAM counts. TAMs are associated with vascular invasion and nodal metastasis, and sTAM and tTAM counts are correlated with overall survival, suggesting they could be used as prognostic indicators in canine mammary carcinomas.

Immunoliposomes bearing lymphocyte activation gene 3 fusion protein and P5 peptide: A novel vaccine for breast cancer.

LAG3-Ig as an immune adjuvant has elicited potent anti-tumor immune responses in several preclinical and clinical studies, but the full potential immunostimulatory of LAG3-Ig has yet to be achieved. We hypothesized that by anchoring LAG3-Ig to the surface of liposomes, the adjuvant activity of LAG3-Ig could be improved. We also investigated the immunotherapy by co-delivery of liposome-coupled LAG3-Ig and P5 tumor antigen in mice model of TUBO breast cancer. We prepared and characterized novel PEGylated liposomes bearing surface conjugated LAG3-Ig and P5. Consistent with our hypothesis, liposomes-conjugated LAG3-Ig via multivalent binding to MHC class II molecules exerted immunostimulatory of LAG3-Ig and markedly induced maturation of dendritic cells more efficiently than free LAG3-Ig. LAG3-Ig-P5-immunoliposomes effectively elicited protective anti-tumor responses more than locally injected soluble LAG3-Ig + P5. The higher percentage of CD4+ and CD8+ T cells in the spleen and more rapid and pronounced infiltration of these effector cells into the site of the tumor were seen following immunoliposome therapy. Finally, anti-tumor immunity induced by LAG3-Ig-P5-immunoliposomes translated into the more tumor regression and prolonged survival of treated mice, compared to soluble immunotherapy. Taken together, our findings suggest that LAG3-Ig-P5-immunoliposomes can be considered as a valuable candidate for developing a liposome-based therapeutic cancer vaccine in treating HER2/ neu+ breast cancer patients.

Significant association of serum autoantibodies to TYMS, HAPLN1 and IGFBP5 with early stage canine malignant mammary tumours.

Canine mammary tumours (CMTs) are the most prevalent neoplasms in female dogs. Despite the high incidence of such tumours, a lack of easily accessible biomarkers still impedes early diagnosis of malignant CMTs. Herein we identify thymidylate synthetase (TYMS), hyaluronan and proteoglycan link protein 1 (HAPLN1) and insulin-like growth factor-binding protein 5 (IGFBP5) as CMT antigens eliciting corresponding autoantibodies in CMT cases. We establish enzyme-linked immunosorbent assays (ELISAs) to detect autoantibodies to TYMS (TYMS-AAb), HAPLN1 (HAPLN1-AAb) and IGFBP5 (IGFBP5-AAb) in sera from 81 dogs with malignant CMTs (41 in Stage I), 24 with benign CMTs and 35 healthy controls. Levels of all the three autoantibodies are elevated in the malignant group compared with the healthy or the benign group; notably, the elevated autoantibody levels significantly correlate with the stage-I CMTs. For discriminating malignant CMTs from healthy control, the area under curve (AUC) of TYMS-AAb is 0.694 with specificity of 82.9% and sensitivity of 50.6%. The AUC of utilising HAPLN1-AAb for distinguishing the stage-I CMTs from healthy controls is 0.711 with specificity of 77.1% and sensitivity of 58.5%. In differentiating malignant CMTs from the benign, the AUC of IGFBP5-AAb reaches 0.696 with specificity of 70.8% and sensitivity of 67.9%, and a combination of IGFBP5-AAb and TYMS-AAb increases the AUC to 0.72. Finally, the AUC of combined HAPLN1-AAb and IGFBP5-AAb in discriminating the stage-I CMTs from the benign achieves 0.731. Collectively, this study highlights a significant association of the three serum autoantibodies with early stage malignant CMTs.

Differential Immune Modulation With Carbon-Ion Versus Photon Therapy.

PURPOSE: Radiation therapy (RT) modulates the immune characteristics of the tumor microenvironment (TME). It is not known whether these effects are dependent on the type of RT used. METHODS AND MATERIALS: We evaluated the immunomodulatory effects of carbon-ion therapy (CiRT) compared with biologically equivalent doses of photon therapy (PhRT) on solid tumors. Orthotopic 4T1 mammary tumors in immunocompetent hosts were treated with CiRT or biologically equivalent doses of PhRT. Seventy-two hours after RT, tumors were harvested and the immune characteristics of the TME were quantified by flow cytometry and multiplex cytokine analyses. RESULTS: PhRT decreased the abundance of CD4+ and CD8+ T cells in the TME at all doses tested, with compensatory increases in proliferation. By contrast, CiRT did not significantly alter CD8+ T-cell infiltration. High-dose CiRT increased secretion of proinflammatory cytokines by tumor-infiltrating CD8+ T cells, including granzyme B, IL-2, and TNF-α, with no change in IFN-γ. Conversely, high-dose PhRT increased CD8+ T-cell secretion of IFN-γ only. At most of the doses studied, PhRT increased proliferation of immunosuppressive regulatory T cells; this was only seen with high-dose CiRT. Cytokine analyses of bulk dissociated tumors showed that CiRT significantly increased levels of IFN-γ, IL-2, and IL-1ß, whereas PhRT increased IL-6 levels alone. CONCLUSIONS: At low doses, lymphocytes differ in their sensitivity to CiRT compared with PhRT. Unlike PhRT, low-dose CiRT is generally lymphocyte-sparing. At higher doses, CiRT is a more potent inducer of proinflammatory cytokines and merits further study as a modulator of the immunologic characteristics of the TME.

Dual roles of neutrophils in metastatic colonization are governed by the host NK cell status.

The role of neutrophils in solid tumor metastasis remains largely controversial. In preclinical models of solid tumors, both pro-metastatic and anti-metastatic effects of neutrophils have been reported. In this study, using mouse models of breast cancer, we demonstrate that the metastasis-modulating effects of neutrophils are dictated by the status of host natural killer (NK) cells. In NK cell-deficient mice, granulocyte colony-stimulating factor-expanded neutrophils show an inhibitory effect on the metastatic colonization of breast tumor cells in the lung. In contrast, in NK cell-competent mice, neutrophils facilitate metastatic colonization in the same tumor models. In an ex vivo neutrophil-NK cell-tumor cell tri-cell co-culture system, neutrophils are shown to potentially suppress the tumoricidal activity of NK cells, while neutrophils themselves are tumoricidal. Intriguingly, these two modulatory effects by neutrophils are both mediated by reactive oxygen species. Collectively, the absence or presence of NK cells, governs the net tumor-modulatory effects of neutrophils.

Tumors Resistant to Checkpoint Inhibitors Can Become Sensitive after Treatment with Vascular Disrupting Agents.

Immune therapy improves cancer outcomes, yet many patients do not respond. This pre-clinical study investigated whether vascular disrupting agents (VDAs) could convert an immune unresponsive tumor into a responder. CDF1 mice, with 200 mm3 C3H mammary carcinomas in the right rear foot, were intraperitoneally injected with combretastatin A-4 phosphate (CA4P), its A-1 analogue OXi4503, and/or checkpoint inhibitors (anti-PD-1, PD-L1, or CTLA-4 antibodies), administered twice weekly for two weeks. Using the endpoint of tumor growth time (TGT5; time to reach five times the starting volume), we found that none of the checkpoint inhibitors (10 mg/kg) had any effect on TGT5 compared to untreated controls. However, CA4P (100 mg/kg) or OXi4503 (5-50 mg/kg) did significantly increase TGT5. This further significantly increased by combining the VDAs with checkpoint inhibitors, but was dependent on the VDA, drug dose, and inhibitor. For CA4P, a significant increase was found when CA4P (100 mg/kg) was combined with anti-PD-L1, but not with the other two checkpoint inhibitors. With OXi4503 (50 mg/kg), a significant enhancement occurred when combined with anti-PD-L1 or anti-CTLA-4, but not anti-PD-1. We observed no significant improvement with lower OXi4503 doses (5-25 mg/kg) and anti-CTLA-4, although 30% of tumors were controlled at the 25 mg/kg dose. Histological assessment of CD4/CD8 expression actually showed decreased levels up to 10 days after treatment with OXi4503 (50 mg/kg). Thus, the non-immunogenic C3H mammary carcinoma was unresponsive to checkpoint inhibitors, but became responsive in mice treated with VDAs, although the mechanism remains unclear.

Chemokine gene expression influences metastasis and survival time of female dogs with mammary carcinoma.

Chemokines are signaling proteins secreted by immune cells which regulate leukocyte trafficking. The aberrant expression of chemokines and their receptors by neoplastic cells influences the behaviour of many human cancers. This study evaluated gene-expression of the chemokines: CCL5, CXCL10, CXCL12 and the chemokine receptors: CXCR3, CXCR4, CXCR7, CCR4, CCR9 in 41 histologically-malignant, outcome-known, canine mammary tumours. These chemokines and chemokine receptors were selected as all were previously shown to influence the behaviour of human breast cancers. The expression of chemokines CCL5 and CXCL12 were significantly higher in tumours which subsequently metastasised than tumours that did not metastasise (p < 0.05). Increased expression of these chemokines was also correlated with shorter survival times of the dogs (CCL5: rs = -0.40, p = 0.02, CXCL12: rs = -0.40, p = 0.03) while CCL5 was independently prognostic of survival times (p = 0.026). A significantly higher proportion of tumours that subsequently metastasised expressed CXCR3 (p = 0.037), CXCR4 (p = 0.026), CXCR7 (p = 0.025) and CCR9 (p = 0.039) receptors while the survival times of the dogs with tumours that expressed CXCR4 (p = 0.045) and CCR9 (p = 0.039) receptors were significantly shorter than dogs with tumours that did not express these receptors. Chemokine and chemokine receptor gene-expression has not been previously correlated with disease outcome of canine mammary tumours. These findings indicate that altered expression of chemokines and their receptors influences the behaviour of canine mammary tumours suggesting a potential role of them as prognostic markers or therapeutic targets.

Targeting Inhibition of Foxp3 by MMP2/9 Sensitive Short Peptide Linked P60 Fusion Protein 6(P60-MMPs) to Enhance Antitumor Immunity.

Regulatory T-cells (Tregs) play an important role in tumor immunosuppressive network, thus Tregs-targeted strategy is expected to enhance antitumor immunity and improve the effect of immunotherapy. Short peptide P60 can bind to the forkhead box protein P3 (Foxp3), a crucial transcriptional regulator for the development and inhibitory function of Tregs, and inhibit Foxp3 nuclear translocation in Tregs. However, its treatment effect in cancer is limited due to nonspecificity. Therefore, realizing the specific delivery of P60 in tumor microenvironment will greatly facilitate its Treg-suppressing effect for tumor therapeutics. Herein, utilizing the unique matrix metallase protease 2/9 (MMP2/9) overexpressing feature in tumor tissues, a fusion protein 6(P60-MMPs) containing six segments of P60 linked by MMP2/9-sensitive peptides is constructed for antitumor targeting immunotherapy. The fusion protein 6(P60-MMPs) specifically degrades into short peptide P60 in tumor, and then binds to Foxp3 to inhibit Foxp3 nuclear translocation in Tregs, thus impairing Tregs' activity. This fusion protein efficiently inhibits murine breast cancer 4T1 transplanted tumor growth and decreases lung metastasis through down-regulating tumor-infiltrated Tregs and up-regulating CD8+ T cells in tumor tissue. The study develops a Treg-targeted anticancer fusion protein with effective therapeutic activity, suggesting its potential in clinical translation.

CDDO-Me Alters the Tumor Microenvironment in Estrogen Receptor Negative Breast Cancer.

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.