Myeloid-derived Suppressor Cells In Spleen Research Articles

Protective effect of Cyclo(His-Pro) on peritoneal fibrosis through regulation of HDAC3 expression.

Peritoneal dialysis is a common treatment for end-stage renal disease, but complications often force its discontinuation. Preventive treatments for peritoneal inflammation and fibrosis are currently lacking. Cyclo(His-Pro) (CHP), a naturally occurring cyclic dipeptide, has demonstrated protective effects in various fibrotic diseases, yet its potential role in peritoneal fibrosis (PF) remains uncertain. In a mouse model of induced PF, CHP was administered, and quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry was employed to identify PF-related protein signaling pathways. The results were further validated using human primary cultured mesothelial cells. This analysis revealed the involvement of histone deacetylase 3 (HDAC3) in the PF signaling pathway. CHP administration effectively mitigated PF in both peritoneal tissue and human primary cultured mesothelial cells, concurrently regulating fibrosis-related markers and HDAC3 expression. Moreover, CHP enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) while suppressing forkhead box protein M1 (FOXM1), known to inhibit Nrf2 transcription through its interaction with HDAC3. CHP also displayed an impact on spleen myeloid-derived suppressor cells, suggesting an immunomodulatory effect. Notably, CHP improved mitochondrial function in peritoneal tissue, resulting in increased mitochondrial membrane potential and adenosine triphosphate production. This study suggests that CHP can significantly prevent PF in peritoneal dialysis patients by modulating HDAC3 expression and associated signaling pathways, reducing fibrosis and inflammation markers, and improving mitochondrial function.

The effects of high plasma levels of Aβ1-42 on mononuclear macrophage in mouse models of Alzheimer’s disease

More and more evidences are proving that microglia play a crucial role in the pathogenesis of Alzheimer’s disease (AD) and the plasma Aβ1-42 levels significantly increased 15 years before the onset of dominantly inherited AD. However, the effects of high plasma levels of Aβ1-42 on mononuclear macrophage, the peripheral counterparts of microglia, remain unclear. In the present study, we used APP/PS1 transgenic (Tg) mice and a parabiotic model of wild type (Wt) mice and Tg mice (Parabiotic Wt-Tg, Pa (Wt-Tg)) to investigate the effects of high plasma levels of Aβ1-42 on peripheral mononuclear macrophage. Our results showed that in the early stage of Tg mice (7 months) and Pa (Wt-Tg) mice (4 months), the proportions of pro-inflammatory macrophages in peritoneal cavity, myeloid derived suppressor cells (MDSCs) in spleen, granulocyte-monocyte progenitors (GMPs) in bone marrow, and the plasma levels of interleukin-6 (IL-6) were significantly decreased. While the proportions of pro-inflammatory macrophages, MDSCs, GMPs, and the plasma levels of IL-6 and tumor necrosis factor (TNF)-α, as well as the numbers of bone marrow-derived macrophages (BMDMs) in mice brain were increased in the late stage of Tg mice (11 months) and Pa (Wt-Tg) mice (8 months). In addition, the proportions of monocytes in spleen and the proliferation of bone marrow cells (BMCs) were enhanced consistently, and the phagocytic function of macrophages kept stably after high plasma levels of Aβ1-42 sustaining stimulation. These results demonstrated that high plasma levels of Aβ1-42 play a biphasic regulating role at different stages of the disease, namely inhibiting effects on peripheral pro-inflammatory macrophages in the early stage of AD model, while promoting effects in the late stage of AD model. The mechanism behind this may be associated with their effects on MDSCs in spleen and myeloid progenitor cells in bone marrow. Therefore, intervening the effects of plasma Aβ1-42 on pro-inflammatory macrophages might offer a new therapeutic approach to AD.

Thyroid status regulates the tumor microenvironment delineating breast cancer fate.

The patient's hormonal context plays a crucial role in the outcome of cancer. However, the association between thyroid disease and breast cancer risk remains unclear. We evaluated the effect of thyroid status on breast cancer growth and dissemination in an immunocompetent mouse model. For this, hyperthyroid and hypothyroid Balb/c mice were orthotopically inoculated with triple-negative breast cancer 4T1 cells. Tumors from hyperthyroid mice showed an increased growth rate and an immunosuppressive tumor microenvironment, characterized by increased IL-10 levels and decreased percentage of activated cytotoxic T cells. On the other hand, delayed tumor growth in hypothyroid animals was associated with increased tumor infiltration of activated CD8+ cells and a high IFNγ/IL-10 ratio. Paradoxically, hypothyroid mice developed a higher number of lung metastasis than hyperthyroid animals. This was related to an increased secretion of tumor CCL2 and an immunosuppressive systemic environment, with increased proportion of regulatory T cells and IL-10 levels in spleens. A lower number of lung metastasis in hyperthyroid mice was related to the reduced presence of mesenchymal stem cells in tumors and metastatic sites. These animals also exhibited decreased percentages of regulatory T lymphocytes and myeloid-derived suppressor cells in spleens but increased activated CD8+ cells and the IFNγ/IL-10 ratio. Therefore, thyroid hormones modulate the cellular and cytokine content of the breast tumor microenvironment. A better understanding of the mechanisms involved in these effects could be a starting point for the discovery of new therapeutic targets for breast cancer.

All-trans-retinoic acid restores CD4+ T cell response after sepsis by inhibiting the expansion and activation of myeloid-derived suppressor cells

BackgroundPatients are susceptible to immunosuppression in late-stage of sepsis, in which myeloid-derived suppressor cells (MDSCs) is an important contributor. This study aims to investigate whether all-trans-retinoic acid (ATRA), which has been proved to inhibit MDSCs generation in cancer, will ameliorate sepsis-induced immuno-suppression through modulating MDSCs. MethodsA clinically relevant “two-hit’’ model of sepsis, the cecal ligation and puncture (CLP) model and secondary pneumonia model, were established in mice. The effects of ATRA on the mortality, the bacterial burden, the expansion and activity of CLP-induced MDSCs, as well as the function of CD4+ T cells were evaluated. ResultsIn CLP model, ATRA was found to reduce frequency of MDSCs in spleen of mice and inhibit activity of MDSCs by regulating the generation and activity of arginase-1 and iNOS, and the secretion of immune-supressive cytokines. ATRA administration eventually reduced mortality of secondary infection by Legionella pneumophila in CLP-surviving mice, which might be associated with the restoration of CD4+ T cells proliferating and secreting activity. ConclusionATRA can restore CD4+ T cells dysfunction in sepsis by modulating the expansion and function of MDSCs and therefore provides a potential therapy that targets the immunosuppressive state of sepsis.

Liver X receptor agonist GW3965 protects against sepsis by promoting myeloid derived suppressor cells apoptosis in mice

AimsImmunosuppressive myeloid-derived suppressor cells (MDSCs) continuously expand and lead to poor outcome during sepsis. The activation of liver X receptor (LXR) can mitigate sepsis-induced liver and myocardial damage. This study aims to determine whether LXR plays a protective role in sepsis by regulating MDSCs. Main methodsCecal ligation and puncture(CLP)was used to induce sepsis in mice. The mice were then treated with LXR agonist GW3965 (3 mg/kg) or vehicle 1 h, 6 h, 12 h, 24 h, 48 h, 72 h postoperatively. The effect of LXR on the survival rate and multi-organ injury induced by sepsis was evaluated by survival analysis, histological staining, biochemical analysis and ELISAs. The percentages of MDSCs and T cells were detected using flow cytometry. The mRNA expressions of LXR and ATP-binding cassette transporter A1 (ABCA1) were measured using real-time quantitative PCR (RT-qPCR). ABCA1 protein level was determined using immunofluorescence staining. Key findingsLXR agonist GW3965 treatment improved the survival of septic mice, accompanied by reduced multi-organ injury and a decreased level of inflammatory cytokines. Furthermore, GW3965 treatment decreased MDSCs abundance in spleen by boosting the apoptosis of spleen MDSCs, therefore ameliorating their immunosuppressive activity. Meanwhile, bacteria clearance in tissues was enhanced after the GW3965 administration in septic mice. Mechanistically, GW3965 activated LXRβ and its downstream target ABCA1 to initiate the apoptosis of spleen MDSCs. SignificanceThese findings provide new insights into the relationship between LXR and MDSCs in sepsis, thus revealing a potentially effective approach to target the immunosuppression of sepsis.

Renal cancer-derived exosomes induce tumor immune tolerance by MDSCs-mediated antigen-specific immunosuppression

BackgoundAlthough Myeloid-derived suppressor cells (MDSCs) have a prominent ability to suppress the immune responses of T lymphocytes and propel tumor immune escape, a lack of profound systemic immunesuppression in tumor-bearing mice and tumor patients. The underlying mechanism of these remains unclear.MethodsFor this purpose, renal cancer-derived exosomes (RDEs) were first labeled with PKH67 and been observed the internalization by MDSCs. Flow cytometry analysis showed the proportion and activity change of MDSCs in spleen and bone marrow induced by RDEs. Further, western blot experiments were used to verify triggered mechanism of MDSCs by RDEs. Finally, proliferation and cytotoxicity of cytotoxic T lymphocytes (CTLs) co-cultured with MDSCs in vitro and a series of experiments in vivo were performed to demonstrate the specific inhibitory effect of RDEs-induced MDSCs.ResultsThis study suggested that RDEs crucially contributed to presenting antigenic information, activating and driving specific immunosuppressive effect to MDSCs. HSP70, which is highly expressed in RDEs, initiate this process in a toll like receptor 2 (TLR2)-dependent manner. Importantly, RDEs-induced MDSCs could exert an antigen-specific immunosuppression effect on CTL and specific promote renal tumors-growth and immune escape in consequence.ConclusionThe immunosuppression mediated by MDSCs which is induced by RDEs is antigen-specific. HSP70, which is highly expressed in RDEs, plays a pivotal role in this process. Targeted abrogating the function of MDSCs, or eliminating the expression of HSP70 in exosomes, or blocking the crosstalk between them provides a new direction and theoretical support for future immunotherapy.CXGDvbic__A2y3SzVzAv3wVideo abstractGraphical abstract

A novel methodology of the myeloid-derived suppressor cells (MDSCs) generation with splenic stroma feeder cells

Myeloid-derived suppressor cells (MDSCs) are a significant obstacle for immunotherapy of cancer. It is of great clinical relevance to study the mechanism of MDSCs accumulation in mouse spleens and establish a stable method to obtain high-purity MDSCs in vitro for further research. Here, we established a new method for amplifying a large number of highly pure MDSCs in vitro. To mimic the microenvironment of MDSCs development in vivo, mouse splenic stroma feeder cells and serum-free medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) were used to induce myeloid precursors in mouse bone marrow cells, which differentiate into MDSCs. Development and immunological functions of the cells were monitored both in vivo and in vitro. A total of 4 × 108 MDSCs could be obtained from the bone marrow from one mouse, the ratio of CD11b+Gr-1+ MDSCs could reach 93.8% ± 3.3% after nine days of culture in vitro. Cultured MDSCs maintained a similar immunophenotype with MDSCs found in tumor-bearing mice. Colony forming assay in vitro and in vivo demonstrated that these were myeloid precursor cells. These cells generated high levels of reactive oxygen species and arginase 1 to prevent proliferation of CD8+ T cells in vitro. These also increased regulatory T (Treg) cells in blood while promoting the growth of lymphoma in vivo. In addition, cultured MDSCs effectively inhibited acute graft-versus-host disease (aGVHD). Our findings suggest that mouse splenic stroma plays an important role in the generation of MDSCs and represent a preliminary mechanism for the accumulation of MDSCs in spleens, and thereby lay the foundation for basic research and the clinical application of MDSCs.

Anti-HER2/Neu antibody therapy can reduce the immunosuppressive activity of MDSCs in breast tumor model.

e14181 Background: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell populations that play a critical role in tumor associated immune suppression. MDSCs consist of immature cells with myeloid lineage markers CD11b and Gr1 in mice and accumulate in large numbers in tumor bearing mice including HER2/neu+ breast cancers. This study was conducted to test whether anti-neu antibody treatment can affect on MDSC populations and immunosuppressive functions. Methods: Female BALB/c mice(5–6weeks of age) were used. The TUBO mammary carcinoma cell line was cloned from a spontaneous mammary tumor in a BALB Neu Tg mouse, as cell lines and reagents. Tumor-infiltrating lymphocyte (TIL) populations were evaluated by flow cytometry. The RT2 Profiler PCR array was used for cytokine expression analysis according to the manufacturer’s instructions. Results: Total number of MDSC in tumor and spleen were decreased within 3 days after anti-neu antibody treatment, but percentage of MDSC in tumor (day2) was reduced more quickly than percentage of MDSC in spleen (day3). The relative populations of MDSC in CD45.2+ cells were significantly decreased such as MO-MDSC. Immunosuppressive activity of MDSC was also reduced by anti-neu antibody treatment. MDSC reduced by anti-neu antibody was due to inhibition of the immunosuppressive factors Arg1 and IDO. In addition, combination therapy with 5-FU may enhance MDSC reduction. Conclusions: MDSC can be a potential therapeutic target in anti-neu antibody treated therapeutic model and the effect of anti-neu antibody could be further improved using other therapeutic treatment to target MDSC.

Abstract 1748: Head and neck cancer-derived exosomes facilitate carcinogenesis in the murine 4NQO chemically induced oropharyngeal cancer

Abstract Purpose: An increasing body of evidence suggests that tumor-derived exosomes (TEX) promote tumor formation and progression. This study aims to show that exosomes produced by the SCCVII murine tumor cell line promote progression of pre-malignant oropharyngeal lesions to malignant tumors. The 4-Nitroquinoline 1-oxide (4NQO) tumor model is chemically induced in immunocompetent mice. It recapitulates all the histological stages and molecular changes associated with of oral carcinogenesis described for human head and neck squamous cell carcinoma (HNSCC). Experimental design: Exosomes were isolated from supernatant of a murine as well as human HNSCC cell-lines by mini size-exclusion chromatography (miniSEC). Morphology, number, protein and the molecular cargo of the recovered exosomes were determined. Exosome-mediated in vitro inhibition of lymphocyte proliferation and induction of CD8+ T-cell apoptosis were demonstrated. C57BL/6 mice were treated with 4NQO supplied in drinking water (100ug/ml) for 16 weeks and all developed oral and esophageal pre-malignant lesions. Exosomes were administered via a single tail-vein or retro-orbital injection at week 18. Tumor sizes and overall tumor burden per mouse were measured with a caliper. Proliferation indexes of individual tumors were measured by immunohistochemistry. Splenocytes were harvested and were analyzed for the presence and frequency of myeloid derived suppressor cells (MDSC) and Tregs, and for in vitro responsiveness of T cells to exogenous activators. Results: SCCVII-derived exosomes were found to carry an immunosuppressive cargo, including CD39, CD73, PD-L1, FasL and COX-2. One-time 90ug injections (IV) of exosomes to groups of >8 mice induced a statistically significant increase in the number of developing tumors and in the overall tumor burden per mouse, relative to control mice injected with PBS. The average number of tumors per mouse was 6.3 versus 3.6, respectively (p < 0.005). TEX derived from human HNSCC cell lines also induced a greater though not statistically significant number of tumors in 4NQO-treated mice. The MDSC frequency was increased in the exosome-injected mice relative to controls. The Treg frequency was not altered. Conclusions: The data demonstrate that the delivery of Head and Neck Cancer-derived exosomes enhanced carcinogenesis of the 4NQO-induced tumors in mice. Augmented carcinogenesis was associated with systemic immunosuppression and increases in the frequency of MDSCs in spleens of mice treated with exosomes. Citation Format: Beatrice M. Razzo, Chang-Sook Hong, Kellsye P. Fabian, Nils Ludwig, Priyanka Sharma, Walter J. Storkus, Theresa L. Whiteside. Head and neck cancer-derived exosomes facilitate carcinogenesis in the murine 4NQO chemically induced oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1748.

Myeloid-Derived Suppressor Cells Induce Podocyte Injury Through Increasing Reactive Oxygen Species in Lupus Nephritis

The expansion of myeloid-derived suppressor cells (MDSCs) has been documented in murine models and patients with lupus nephritis (LN), but the exact role of MDSCs in this process remains largely unknown. In this study, we investigated whether MDSCs are involved in the process of podocyte injury in the development of LN. In toll-like receptor-7 (TLR-7) agonist imiquimod-induced lupus mice, we found the severe podocyte injury in glomeruli of lupus mice and significant expansion of MDSCs in spleens and kidneys of lupus mice. The function of TLR-7 activated MDSCs was enhanced including the increased generation of reactive oxygen species (ROS) in vivo and in vitro. Moreover, the ROS production of MDSCs induced podocyte injury through activating the p-38MAPK and NF-kB signaling. Furthermore, we verified that podocyte injury was indeed correlated with expansion of MDSCs and their ROS secretion in LN of pristane-induced lupus mice. These findings first indicate that the podocyte injury in LN was associated with the increased MDSCs in kidney and MDSCs may be a promising therapeutic target of LN in the future.

Phenformin Inhibits Myeloid-Derived Suppressor Cells and Enhances the Anti-Tumor Activity of PD-1 Blockade in Melanoma

Biguanides, such as the diabetes therapeutics metformin and phenformin, have shown antitumor activity both invitro and invivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin selectively inhibits granulocytic myeloid-derived suppressor cells in spleens of tumor-bearing mice and exvivo. Phenformin induces production of reactive oxygen species in granulocytic myeloid-derived suppressor cells, whereas the antioxidant N-acetylcysteine attenuates the inhibitory effects of phenformin. Co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN-null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8+ T-cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of myeloid-derived suppressor cells. Our findings show a selective, inhibitory effect of phenformin on granulocytic myeloid-derived suppressor cell-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma.

Distribution and differentiation of myeloid-derived suppressor cells after fluid resuscitation in mice with hemorrhagic shock.

To investigate the distribution and differentiation of myeloid-derived suppressor cells (MDSCs) in hemorrhagic shock mice, which are resuscitated with normal saline (NS), hypertonic saline (HTS), and hydroxyethyl starch (HES). BALB/c mice were randomly divided into control, NS, HTS, and HES resuscitation groups. Three subgroups (n=8) in each resuscitation group were marked as 2, 24, and 72 h. Flow cytometry was used to detect the MDSCs, monocytic MDSCs (M-MDSCs), and granulocytic/neutrophilic MDSCs (G-MDSCs) in peripheral blood nucleated cells (PBNCs), spleen single-cell suspension, and bone marrow nucleated cells (BMNCs). The MDSCs in BMNCs among three resuscitation groups were lower 2 h after shock, in PBNCs of the HTS group were higher, and in spleen of the NS group were lower (all P<0.05 vs. control). The M-MDSC/G-MDSC ratios in PBNCs of the HTS and HES groups were lower (both P<0.05 vs. control). At 24 h, the MDSCs in PBNCs of the NS and HTS groups were higher, while the spleen MDSCs in the HTS group were higher (all P<0.05 vs. control). The M-MDSC/G-MDSC ratios were all less in PBNCs, spleen, and BMNCs of the NS and HTS groups, and were lower in BMNCs of the HES group (all P<0.05 vs. control). At 72 h, the elevated MDSCs in PBNCs were presented in the HTS and HES groups, and in spleen the augment turned up in three resuscitation groups (all P<0.05 vs. control). The inclined ratios to M-MDSC were exhibited in spleen of the NS and HTS groups, and in PBNCs of the NS group; the inclination to G-MDSC in BMNCs was shown in the HES group (all P<0.05 vs. control). HTS induces the earlier elevation of MDSCs in peripheral blood and spleen, and influences its distribution and differentiation, while HES has a less effect on the distribution but a stronger impact on the differentiation of MDSCs, especially in bone marrow.

Inhibition of curcumin on myeloid-derived suppressor cells is requisite for controlling lung cancer

Lung cancer remains the leading cause of cancer mortality. Myeloid-derived suppressor cells (MDSCs) are potent immune-suppressive cells and present in most cancer patients. Recently, several studies have shown that curcumin inhibits the expansion of MDSCs in some cancers. However, it is not clear how curcumin modulates the suppressive function of MDSCs, and whether curcumin achieves anti-tumor effects via regulating the expansion of MDSCs in lung cancer. Here, our results showed that curcumin significantly inhibited tumor growth in a Lewis lung carcinoma (LLC) isogenic tumor model. Curcumin reduced the accumulation of MDSCs in spleen and tumor tissue in LLC isogenic model. And curcumin promoted the maturation and differentiation of MDSCs in tumor tissue. Notably, curcumin inhibited the expression level of immune suppressive factors of MDSCs, arginase-1 (Arg-1) and ROS, in purified MDSCs from tumor tissue in vivo. Expectedly, curcumin also inhibited the immunosuppressive function of isolated MDSCs from tumor tissue and spleen of tumor bearing mice in vitro. Moreover, curcumin decreased the level of IL-6 in the tumor tissue and serum from LLC-bearing mice. Taken together, curcumin indeed possesses anti-cancer effect and inhibits the accumulation and function of MDSCs. And curcumin reduces the level of IL-6 in tumor-bearing mice to impair the expansion and function of MDSCs. These results suggest that inhibition of MDSCs in tumor is requisite for controlling lung cancer.

Abstract 1413: Treatment of mouse liver and brain colon cancer metastases with Toca 511 and 5-fluorocytosine for intratumoral production of 5-fluorouracil leads to increased survival, induction of antitumor immune responses, and reduction of MDSC

Abstract Approximately 50% of patients with colorectal cancer (CRC) develop metastases (mCRC) during the course of disease, with liver the most frequent site. Standard treatment for mCRC is 5-fluorouracil (5-FU) based combination therapy, with median survival now &amp;gt;20 months. As a result of prolonged survival with metastatic disease, incidence of brain metastases from colorectal cancer is increasing. Approved immunotherapeutic agents have had limited effect in mCRC except in the subset of subjects (3-6%) with deficient mismatch repair. Recent studies suggest myeloid-derived suppressor cells (MDSC) contribute to cancer immune evasion by suppressing T cell anti-tumor functions and modulating innate immune responses. We are pursuing a unique approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector, selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) protein. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an oral anti-fungal drug) to 5-FU. Direct tumor cytotoxicity and extended immunotherapeutic effects have been reported using this approach. Toca 511 and Toca FC (extended release 5-FC) is under investigation in subjects with primary brain tumors, delivered intratumorally (NCT01156584), by injection into the resection bed (NCT01470794, NCT02414165), or intravenously (NCT01985256). We tested the intravenous (IV) approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci in the liver. IV delivery of Toca 511 followed by courses of 5-FC resulted in tumor response, improved survival, and immune mediated inhibition of rechallenge with tumor. IV delivery resulted in expression of vector encoded transgene in tumor foci but not in adjacent normal liver. Similar published results have been obtained in a number of brain tumor models with Toca 511+5-FC including a CT26 colon carcinoma brain metastasis model. We further investigated the effect of Toca 511+5-FC treatment on CRC tumor-induced MDSC in the CT-26 brain metastasis model. Intracranial cell implantation and intratumoral Toca 511 vector injections were followed by one course of 5-FC. Expansion of MDSC occurred in brain tumors and spleens of tumor bearing animals. A significant decrease in MDSC in spleens and tumors was observed with Toca 511+5-FC treatment compared to control (p = 0.03, p&amp;lt;0.0001; respectively). The results reported here support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665). Citation Format: Maria Rodriguez-Aguirre, Kader Yagiz, Fernando Lopez Espinoza, Daniel Mendoza, Tiffany T. Huang, Carlos Ibanez, Harry E. Gruber, Douglas J. Jolly, Joan Robbins. Treatment of mouse liver and brain colon cancer metastases with Toca 511 and 5-fluorocytosine for intratumoral production of 5-fluorouracil leads to increased survival, induction of antitumor immune responses, and reduction of MDSC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1413.

205. Cytotoxic and Immunotherapeutic Effects of Toca 511 and 5-Fluorocytosine in an Intraperitoneal Model of Metastatic Colorectal Cancer

Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD) gene, selectively replicates and spreads in tumors. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an orally-available anti-fungal drug) to 5-FU (5-fluorouracil), leading to both direct tumor cytotoxicity and extended immunotherapeutic effects in preclinical models. Toca 511 in combination with Toca FC (extended-release 5-FC) recently entered a Phase 2/3 trial (NCT02414165) in patients with recurrent HGG (high grade glioma). Toca 511 is also under investigation delivered via intravenous (IV) infusion followed by injection into the wall of the resection cavity (NCT01985256), followed by Toca FC, in patients with recurrent HGG. IV administration of vectors is minimally invasive, can easily be repeated if desired, and may be applicable to other tumor types including metastatic colorectal cancer (mCRC). Previously we have shown that IV delivery of Toca 511 in a mouse syngeneic mCRC liver model resulted in expression of CD in tumor foci, but not in adjacent normal liver, and followed by courses of 5-FC resulted in direct tumor response, improved survival, and a systemic anti-tumor immune response. Elevated circulating myeloid-derived suppressor cells (MDSC) are associated with advanced disease stages in CRC patients and failure of immunotherapeutic strategies. We further investigated the effect of intralesional administration of Toca 511 with 5-FC treatment on tumor recurrence and immune infiltrates in a model of CRC brain metastases. A significant decrease in MDSC in spleens and tumors was observed with Toca 511 and 5-FC compared to controls, via in situ production of 5-FU (p=0.03, p<0.0001; respectively). Currently, we are investigating the efficacy of Toca 511 and 5-FC in a mouse syngeneic intraperitoneal (IP) model. We identified the optimal delivery method for treatment of IP metastases, and evaluated survival after IV and IP vector delivery followed by courses of 5-FC. Treatment with Toca 511 and 5-FC led to an improved median survival compared to control. Systemic 5-FU has hematological toxicity even at low doses (20 mg/kg) in both naive and CRC tumor-bearing mice which could have an adverse effect on anti-tumor immune responses. We further evaluated Toca 511 and 5-FC treatment compared to systemic 5-FU in terms of efficacy, hematologic toxicity, and induction of anti-tumor immune responses in the IP mCRC model. The effect of Toca 511 and 5-FC compared to systemic 5-FU on MDSC and other immune cell populations will be presented. Our data provides support for the development of Toca 511 and 5-FC as a unique approach targeting both the tumor and the immune system for the treatment of metastatic cancers such as mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665).

Effect of mild hypothermia on the increase of CD11b+ Gr-1+ myeloid-derived suppressor cells induced by lipopolysaccharide in a mouse model of sepsis

PurposeThis study aimed to investigate the influence of mild hypothermia on the number of CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSCs) induced by lipopolysaccharide (LPS) injection in mice with sepsis. MethodsBALB/c mice were administered LPS to establish a mouse model of sepsis. Then, these mice were randomly divided into 3 groups: the mild hypothermia plus LPS group, the normothermia plus LPS group, and the LPS group. The normal control group was injected the same amount of 0.9% sodium chloride solution. The ratio of CD11b+ Gr-1+ MDSCs in the mouse spleen and bone marrow was determined at 6, 12, 24, 48, and 72 hours after LPS injection and after injected 0.9% sodium chloride solution. ResultsCompared with the control group, the number of MDSCs in the spleen in the sepsis group increased gradually, and the difference was significant at 12 hours after injection (P < .01). Moreover, the number of MDSCs was the lowest in the mild hypothermia group, and there was a significant difference than the other 2 groups at 48 hours (P < .01). The number of MDSCs in the bone marrow increased gradually, and the difference between the sepsis and control groups was significant at 24 hours (P < .01). The number of MDSCs in the mild hypothermia group was the lowest, and there was a statistically significant difference than the other 2 groups (P < .05). ConclusionMild hypothermia inhibited the production and accumulation of MDSCs induced by LPS administration in septic mice.

ITOC2 – 033. Influence of interferon-alpha combined with chemo (radio) therapy on immunological parameters in pancreatic adenocarcinoma

Prognosis of patients with carcinoma of the exocrine pancreas is particularly poor. A combination of chemotherapy with immunotherapy could be an option for treatment of pancreatic cancer. The aim of this study was to perform an immunomonitoring of the CapRI-2 patients, and tumour-bearing mice treated with combination of chemo (radio) therapies with interferon-2α. Low doses of interferon-2α led to a decrease in total leucocyte and an increase in monocyte counts. Furthermore, we observed a positive effect of interferon-2α therapy on the dendritic cells and NK cell activation immediately after the first injection. In addition, we recorded an increased amount of interferon-γ and interleukin-10 (IL-10) in the serum following the interferon-2α therapy. These data clearly demonstrate that pancreatic carcinoma patients show an immunomodulatory response to interferon-2α therapy. Analysis of immunosuppressive cells in the Panc02 orthotopic mouse model of pancreatic cancer revealed an accumulation of the myeloid-derived suppressor cells in spleens and tumours of the mice treated with interferon-2α and 5-fluorouracil. The direct effect of the drugs on myeloid-derived suppressor cells was also registered <i>in vitro</i>. These data expose the importance of both immunoactivating and immunosuppressive mechanisms induced by combined chemo-immunotherapy.

Cyclophosphamide and IL-12-transduced DCs enhance the antitumor activity of tumor antigen-stimulated DCs and reduce Tregs and MDSCs number.

A hostile tumor microenvironment, characterized by an abundance of T regulatory cells and myeloid-derived suppressor cells (MDSCs), considerably limits the efficacy of dendritic cell (DC)-based vaccines. The intention of this study was to enhance the antitumor activity of vaccines consisting of bone marrow-derived DCs stimulated with TAg (BMDC/TAg) via single administration of cyclophosphamide and multiple injections of interleukin (IL)-12-transduced DCs (BMDC/IL-12). The combined chemoimmunotherapy was applied in the treatment of mice with subcutaneously (SC) growing, advanced MC38 colon carcinoma. The highest level of tumor growth inhibition, accompanied by high cytotoxic activity of effector cells, and their increased influx into tumor tissue, was observed after application of cyclophosphamide in combination with BMDC/TAg and BMDC/IL-12. The effect was probably associated with the elimination of T regulatory cells from spleens and tumors, but most of all with changes in the number and differentiation stage of MDSCs. After the therapy, the percentage of granulocytic and monocytic MDSCs in spleens was significantly lower than in the control group. Moreover, MDSCs derived from spleens and tumors showed increased expression of MHC class II, which may indicate the higher maturation stage of the myeloid cells as well as their enhanced capacity toward antigen presentation. The obtained data indicate that the optimal composition of antitumor vaccines able to limit the suppressor activity of MDSCs is essential to enhance the elimination of tumor cells and to achieve an optimal therapeutic effect.

Abstract 3616: Hypoxia determines the fate of myeloid cell differentiation by controlling STAT3 activation in tumor site

Abstract The abnormal differentiation of myeloid cells is the key element of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Recent data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. Hypoxia is intricate part of tumor microenvironment. We have found that hypoxia plays a major role in regulation of MDSC differentiation to macrophages in tumor site. Hypoxia caused dramatic reduction in phosphorylated STAT3 in MDSC. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. In our experiments spleen MDSC in tumor-bearing mice had much higher level of STAT3 phosphorylation than their counterparts in tumor site from the same mice. In cancer patients MDSC in tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. In vitro culture of spleen MDSC in hypoxia led to the suppression of STAT3 activation. Furthermore, mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, suggesting that STAT3 is a downstream target for hypoxia driven myeloid cells differentiation in cancer. We found that CD45 protein tyrosine phosphatase was a molecule that regulated STAT3 activation in hypoxia. The activity but not expression of CD45 phosphatase was up-regulated in tumor MDSC as compared to MDSC in spleens. The exposure of spleen MDSC to hypoxia led to the significant increase in CD45 activity. The siRNA mediated silencing of CD45 abrogated the effect of hypoxia on the down-regulation of STAT3 activity in MDSC. This suggests that hypoxia driven up-regulation of CD45 phosphatase activity suppresses STAT3 activation, which in turn plays a key role in differentiation of myeloid cells in tumor site. These unexpected findings may provide rationale for re-evaluation of therapeutic strategies targeting myeloid cells in cancer. Citation Format: Vinit Kumar. Hypoxia determines the fate of myeloid cell differentiation by controlling STAT3 activation in tumor site. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2014-3616

Influence of Interferon-Alpha Combined with Chemo (Radio) Therapy on Immunological Parameters in Pancreatic Adenocarcinoma

Prognosis of patients with carcinoma of the exocrine pancreas is particularly poor. A combination of chemotherapy with immunotherapy could be an option for treatment of pancreatic cancer. The aim of this study was to perform an immunomonitoring of 17 patients with pancreatic cancer from the CapRI-2 study, and tumor-bearing mice treated with combination of chemo (radio) therapies with interferon-2α. Low doses of interferon-2α led to a decrease in total leukocyte and an increase in monocyte counts. Furthermore, we observed a positive effect of interferon-2α therapy on the dendritic cells and NK (natural killer) cell activation immediately after the first injection. In addition, we recorded an increased amount of interferon-γ and IL-10 in the serum following the interferon-2α therapy. These data clearly demonstrate that pancreatic carcinoma patients also show an immunomodulatory response to interferon-2α therapy. Analysis of immunosuppressive cells in the Panc02 orthotopic mouse model of pancreatic cancer revealed an accumulation of the myeloid-derived suppressor cells in spleens and tumors of the mice treated with interferon-2α and 5-fluorouracil. The direct effect of the drugs on myeloid-derived suppressor cells was also registered in vitro. These data expose the importance of immunosuppressive mechanisms induced by combined chemo-immunotherapy.