Antitumor effect of anti-vascular therapy with STING agonist depends on the tumor microenvironment context

IntroductionTargeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models.MethodsOur study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1).ResultsThe use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed.ConclusionThe study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4+, CD8+ and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy

Vasostatin increases oxygenation of B16-F10 melanoma tumors and raises therapeutic efficacy of cyclophosphamide

One of the preconditions of effective anticancer therapy is efficient transfer of the therapeutic agent (chemotherapeutic) to tumor cells. Fundamental barriers making drug delivery and action difficult include underoxygenation, elevated interstitial pressure, poor and abnormal tumor blood vascular network and acidic tumor milieu. In this study we aimed at developing an optimized scheme of administering a combination of an angiogenesis-inhibiting drug (vasostatin) and a chemotherapeutic (cyclophosphamide) in the therapeutic treatment of mice bearing experimental B16-F10 melanoma tumors. We report that the strongest tumor growth inhibition was observed in mice that received two, three or four vasostatin doses in combination with one injection of cyclophosphamide (i.e., V2 + CTX, V3 + CTX or V4 + CTX schemes). Double administration of vasostatin increases oxygenation of B16-F10 tumors. On the other hand, its five-fold administration lowers tumor oxygenation, breaks down tumor vascular network (increasing hypoxia) and leads in consequence to death of cancer cells and appearance of necrotic areas in the tumor. A decreased cyclophosphamide dose in combination with two doses of vasostatin (V2 + CTX scheme) inhibits tumor growth similarly to a larger dose of cyclophosphamide alone

M1-like macrophages change tumor blood vessels and microenvironment in murine melanoma.

Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4+, CD8+ lymphocytes and NK cells. Depletion of TAMs as well as CD8+ lymphocytes and NK cells, but not CD4+ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression

Additional file 1 of The comparison of adipose-derived stromal cells (ADSCs) delivery method in a murine model of hindlimb ischemia

Additional file 1. Materials and Methods: Animals. Figure S1. Separate fluorescence channels of images of M2 macrophages

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart

Depletion of macrophages reduces the effect of combining ENG-based DNA vaccine with IL-12.

<p>One day after inoculating mice (n = 7–8) with B16-F10 cells, the animals were orally vaccinated (three times at one-week intervals). Additionally, on days 9, 11 and 13 following inoculation with cancer cells, IL-12 was injected directly into tumors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191012#sec002" target="_blank">Materials and methods</a>). Moreover, liposomes (‘empty’ liposomes (control) or Clodronate liposomes (Clodr.)) were delivered intraperitoneally at a dose of 10 mg/kg and directly into tumors at a dose of 5 mg/kg, 2 times/week. Depletion of TAMs decreased the growth of control tumors. But in mice treated with combined therapy we observed enhanced tumor growth after TAMs depletion. Photographs were taken on 20^th day of the experiment. Tumors (n = 3) were collected 20 days after challenge and stained with antibodies against CD206 and F4/80. Magnification 20×. *<i>P</i><0.0001, the ANOVA followed by the Tukey’s <i>post hoc</i> test.</p

Effect of combined therapy (ENG vaccine + IL-12) on tumor-immune cells infiltration.

<p>On day 20^th of the combined therapy, mice (n = 9) were sacrificed and tumors were excised for flow analysis. Obtained single-cell suspensions were then used to quantitate the level of CD4⁺, CD8⁺ lymphocytes and NK cells. The percentage of CD4⁺, CD8⁺ lymphocytes and NK cells was determined in total viable cells (representative flow data for CD4⁺, CD8⁺ lymphocytes and NK cell populations). Combined therapy increased tumor-infiltrating CD4⁺ (more than three times), CD8⁺ lymphocytes (more than eight times) as well as NK cells (more than three times) levels compared with control tumors. *<i>P</i><0.0001, the Cochran’s C test; **<i>P</i><0.0001, the Student’s <i>t</i>-test.</p

Depletion of CD8⁺ lymphocytes and NK cells, but not CD4⁺ lymphocytes, reduces the effect of combining ENG-based DNA vaccine with IL-12.

<p>One day after inoculating mice (n = 6–8) with B16-F10 cells, the animals were orally vaccinated (three times at one-week intervals). Additionally, on days 9, 11 and 13 following inoculation with cancer cells, IL-12 was injected directly into tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191012#sec002" target="_blank">Materials and methods</a>). Moreover, monoclonal antibodies (anti-CD8a, anti-CD4 lymphocytes or anti-NK cells) were injected intraperitoneally at a dose of 200 μg per mouse on days -1 (1 day before the first vaccination), 1, 6, 11 and 16. Blood was collected on 10^th day after B16-F10 inoculation for flow analysis. Tumors and spleens were harvested 20 days after challenge and stained with antibodies against CD4, CD8 lymphocytes and NK cells. The percentage of CD4⁺, CD8⁺ lymphocytes and NK cells was determined in total viable lymphoid CD45⁺ cells. Representative flow data for CD4⁺, CD8⁺ lymphocytes and NK cell populations after antibody administration (A). Antibody selectively depleted cells in peripheral blood, spleens and tumors. Depletion of CD8⁺ lymphocytes and NK cells enhanced the growth of tumors in treated mice (more than 3 and 7 times, respectively). But after CD4⁺ lymphocytes depletion we observed more than 3 times decreased tumor growth in mice treated with combined therapy. Photographs were taken on 19^th day of the experiment (B). Magnification 20×. *<i>P</i><0.0001 compared with control (PBS¯) group, **<i>P</i><0.05 compared with NK depletion group, ***<i>P</i><0.01 compared with NK depletion group; the Kruskal-Wallis followed by the <i>post hoc</i> multiple comparisons of rank sums test.</p