Decrease In Myeloid-derived Suppressor Cells Research Articles

Cold Exposure Therapy Sensitizes Nanodrug-Mediated Radioimmunotherapy of Breast Cancer.

Cold exposure (CE) therapy can quickly induce tumor starvation by brown adipose tissue (BAT) thermogenesis. Exploring the combined antitumor mechanism of CE and traditional therapies (such as radiotherapy (RT)) is exciting and promising. In this study, we investigated the effect of CE in combination with nitric oxide (NO) gas therapy on sensitizing tumors to RT and promoting tumor radio-immunotherapy. We first constructed a liposome (SL) loaded with the NO prodrug S-nitroso-N-acetylpenicillamine (SNAP). When SL is injected, the glutathione (GSH) within the tumor region promotes the release of NO from SNAP. Subsequently, the superoxide anion produced by RT reacts with NO to generate peroxynitrite (ONOO-), which has strong oxidative properties and induces cell death. Meanwhile, the mice were exposed to a CE environment of 4 °C. CE-mediated BAT thermogenesis induced tumor starvation, which led to a decrease in ATP and GSH content within the tumor as well as an improvement in the hypoxic microenvironment and a decrease in myeloid-derived suppressor cells. All of the above have promoted the effectiveness of RT and activated the systemic antitumor immunity. In the bilateral tumor experiment, treatment of the primary tumor inhibited the growth of the distant tumor and promoted the infiltration of CD8+ T cells into the tumor. These findings reveal that the synergy of CE, NO gas therapy, and RT could confer high effective anticancer effects, providing possibilities in personalized cancer treatment.

Targeting SIGLEC15 as an emerging immunotherapy for anaplastic thyroid cancer

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer with few effective therapies. Though immunotherapies such as targeting PD-1/PD-L1 axis have benefited patients with solid tumor, the druggable immune checkpoints are quite limited in ATC. In our study, we focused on the anti-tumor potential of sialic acid-binding Ig-like lectins (Siglecs) in ATC. Through screening by integrating microarray datasets including 216 thyroid-cancer tissues and single-cell RNA-sequencing, SIGLEC family members CD33, SIGLEC1, SIGLEC10 and SIGLEC15 were significantly overexpressed in ATC, among which SIGLEC15 increased highest and mainly expressed on cancer cells. SIGLEC15high ATC cells are characterized by high expression of serine protease PRSS23 and cancer stem cell marker CD44. Compared with SIGLEC15low cancer cells, SIGLEC15high ATC cells exhibited higher interaction frequency with tumor microenvironment cells. Further study showed that SIGLEC15high cancer cells mainly interacted with T cells by immunosuppressive signals such as MIF-TNFRSF14 and CXCL12-CXCR4. Notably, treatment of anti-SIGLEC15 antibody profoundly increased the cytotoxic ability of CD8+ T cells in a co-culture model and zebrafish-derived ATC xenografts. Consistently, administration of anti-SIGLEC15 antibody significantly inhibited tumor growth and prolonged mouse survival in an immunocompetent model of murine ATC, which was associated with increase of M1/M2, natural killer (NK) cells and CD8+ T cells, and decrease of myeloid-derived suppressor cells (MDSCs). SIGLEC15 inhibited T cell activation by reducing NFAT1, NFAT2, and NF-κB signals. Blocking SIGLEC15 increased the secretion of IFN-γ and IL-2 in vitro and in vivo. In conclusion, our finding demonstrates that SIGLEC15 is an emerging and promising target for immunotherapy in ATC.

Knock down of APE1 suppressed gastric cancer metastasis via improving immune disorders caused by myeloid-derived suppressor cells

ABSTRACT Gastric cancer is a highly immunogenic malignancy. Immune tolerance facilitated by myeloid-derived suppressor cells (MDSCs) has been implicated in gastric cancer resistance mechanisms. The potential role of APE1 in regulating gastric cancer metastasis by targeting MDSCs remains uncertain. In this study, the plasmid Plxpsp-mGM-CSF was used to induce high expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in GES-1 cells. For tumor transplantation experiments, AGS, AGS+GM-CSF and AGS+GM-CSF-siAPE1 cell lines were established by transfection, followed by subcutaneous implantation of tumor cells. MDSCs, Treg cells, IgG, CD3 and CD8 levels were assessed. Transfection with siAPE1 significantly inhibited tumor growth compared to the AGS+GM-CSF group. APE1 gene knockdown modulated the immune system in gastric cancer mice, characterized by a decrease in MDSCs and an increase in Treg cells, IgG, CD3 and CD8. In addition, APE1 gene knockdown resulted in decreased levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12). Furthermore, APE1 gene knockdown inhibited proliferation, migration and invasion of AGS and MKN45 cells. AGS-GM-CSF cell transplantation increased MDSC levels and accelerated tumor growth, whereas APE1 knockdown reduced MDSC levels, inhibited tumor growth and attenuated inflammatory infiltration in gastric cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach to the prevention and treatment of gastric cancer, providing new insights into its management.

IMN041 is an immunotherapeutic and an effective treatment in mouse xenograft models of pancreatic cancer, renal cancer and triple negative breast cancer

BackgroundiMN013 (5-aza-2',2'-difluorodeoxycytidine), a DNA methyl transferase inhibitor and ribonucleotide reductase inhibitor, and its prodrug iMN041 (3',5'-di-trimethylsilyl-2',2'-difluro-5- azadeoxycytidine), have been shown to be active in mouse xenograft models of hematogenous and solid tumors. In a xenograft of non-small cell lung cancer (NCI-H460), iMN041 treated mice demonstrated a marked inflammatory response upon analysis of tumor histology, which was hypothesized to be mediated by upregulation of natural killer (NK) cells. This study aimed to characterize the antitumor immune responses generated by iMN041 and test the efficacy iMN041 in solid tumors with poor prognosis.MethodsIn the Renca syngeneic mouse model, tumors were harvested following two doses of iMN041 or vehicle control, and analyzed by fluorescent-activated cell sorting for an antitumor immune response. iMN041 was also tested for tumor growth inhibition and animal survival for up to 42 days in xenograft models of pancreatic, renal, and triple negative breast cancer.ResultsTumors from mice implanted with the Renca cell line and treated with iMN041 demonstrated an increase in granzyme B in NK (p = 0.024) and NKT cells (p = 0.004), an increase in the ratios of CD8-T to regulatory T cells (Treg) (p = 0.0026) and CD4-T to Treg cells (p = 0.022) and a decrease in myeloid-derived suppressor cells (p = 0.040), compared to vehicle controls. A significant decrease in MAGE-A positive tumor cells in treated mice, concordant with a proportional decrease in all live tumor cells, suggests that these cells are one of the main targets of the activated immune system. Xenograft models of the triple negative breast cancer cell line DU4475, renal cancer cell lines 786-O and Caki-1, and pancreatic cancer cell lines CFPAC-1 and SW1990, demonstrated significantly lower tumor volumes, and, where there were a sufficient number of events, significantly improved survival in treated mice compared to vehicle control mice.ConclusionsIn mouse cancer models, iMN041 is an effective treatment for solid tumors mediated in part through a unique antitumor immune response.

Polysaccharide isolated from Grifola frondosa eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T cells responses.

Myeloid derived suppressor cells (MDSCs) are known to accumulate in cancer patients and tumor-bearing mice, playing a significant role in promoting tumor growth. Depleting MDSCs has emerged as a potential therapeutic strategy for cancer. Here, we demonstrated that a fungal polysaccharide, extracted from Grifola frondosa, can effectively suppress breast tumorigenesis in mice by reducing the accumulation of MDSCs. Treatment with Grifola frondosa polysaccharide (GFI) leads to a substantial decrease in MDSCs in the blood and tumor tissue, and a potent inhibition of tumor growth. GFI treatment significantly reduces the number and proportion of MDSCs in the spleen, although this effect is not observed in the bone marrow. Further analysis reveals that GFI treatment primarily targets PMN-MDSCs, sparing M-MDSCs. Our research also highlights that GFI treatment has the dual effect of restoring and activating CD8+T cells, achieved through the downregulation of TIGIT expression and the upregulation of Granzyme B. Taken together, our findings suggest that GFI treatment effectively eliminates PMN-MDSCs in the spleen, leading to a reduction in MDSC numbers in circulation and tumor tissues, ultimately enhancing the antitumor immune response of CD8+T cells and inhibiting tumor growth. This study introduces a promising therapeutic agent for breast cancer.

Tucatinib promotes immune activation and synergizes with programmed cell death-1 and programmed cell death-ligand 1 inhibition in HER2-positive breast cancer.

Programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors have poor efficacy in patients with trastuzumab-resistant advanced HER2-positive breast cancer. Tucatinib is a potent, selective anti-HER2 tyrosine kinase inhibitor with proven clinical benefit in the advanced setting in patients with trastuzumab resistance. We investigated if tucatinib can alter the tumor microenvironment and if this could be harnessed for therapeutic efficacy. We investigated the antitumor efficacy and contribution of the immune response of tucatinib using 2 immunocompetent, HER2-positive murine breast cancer models (trastuzumab-sensitive H2N113; trastuzumab-resistant Fo5) and the efficacy of tucatinib with trastuzumab and PD-1 or PD-L1 checkpoint inhibitors. In both models, tucatinib statistically significantly inhibited tumor growth and demonstrated dose-dependent efficacy. Ex vivo analysis by flow cytometry of tumor-infiltrating lymphocytes in mice treated with tucatinib showed increased frequency, higher proliferation, and enhanced effector function of CD8+ effector memory T cells. Tucatinib treatment also increased frequency of CD8+PD-1+ and CD8+TIM3+ T cells, CD49+ natural killer cells, monocytes, and major histocompatibility complex II expression on dendritic cells and macrophages and a decrease in myeloid-derived suppressor cells. Gene expression analysis revealed statistically significant enrichment in pathways associated with immune activation, type I and II interferon response, adaptive immune response, and antigen receptor signaling. In vivo, tucatinib and α-PD-L1 or α-PD-1 demonstrated statistically significantly increased efficacy and improved survival of mice compared with tucatinib alone. Tucatinib modulates the immune microenvironment favorably, and combination treatment with α-PD-L1 or α-PD-1 demonstrated increased efficacy in preclinical HER2-positive tumor models. These findings provide a rationale for investigation of tucatinib and immune checkpoint inhibition in the clinic.

Abstract CT144: Randomized phase II trial of preoperative fulvestrant with or without enzalutamide for ER+/Her2- breast cancer

Abstract Background: Most estrogen receptor alpha positive (ER+) breast cancers (BCs) express androgen receptor (AR) protein, but its function is unclear. High AR relative to ER is associated with endocrine resistance. This randomized phase II trial of neoadjuvant fulvestrant (F) with or without the anti-androgen enzalutamide (E) was designed to determine if the addition of E to F in women with ≥T2 ER+/Her2- primary BC would increase the % patients (pts) with limited residual disease at time of surgery as measured by modified preoperative endocrine predictive index (PEPI). Methods: 4 months of therapy was given prior to surgery (F 500 mg IM weeks 1, 3, 5, 9, and 13) and pts were randomized to receive E 160 mg po daily for 16 weeks, stratified by node status and T-stage. Tumor biopsies were required at study entry and after 4 weeks on therapy (Wk5). PEPI score at time of surgery was the primary endpoint for efficacy. The minimax two-stage design had 80% power with type I error rate of 0.08. Reverse phase phosphoprotein array (RPPA), IHC for ER/PR/AR/GR/Ki67, and Polaris multiplex immunofluorescence (IF) panel for myeloid lineage immune cells were performed. Average signal levels were compared across arms, PEPI score (0, >0), PEPI by arm, and Ki67 (<10%, ≥10%). Comparisons at both baseline and with treatment were considered. Significance was assessed with empirical Bayes moderated t-statistics. Since exploratory, p-values were not adjusted for multiple testing. Results: 69 pts consented; 59 evaluable. 95% completed surgery. PEPI=0 observed in 10 (17%). Of 33 pts on FE arm, 21 had T3/T4 tumors, 91% ER+/PR+, median AR expression 80%, Ki67 15%. Of 26 patients on F arm, 19% had T3/T4 tumors, 96% ER+/PR+, median AR expression 85%, Ki67 10%. PEPI=0 was achieved more frequently on the FE arm (8, 24%) than the F arm (2, 8%) (p=0.16). Toxicity was as expected with endocrine therapy. IHC of baseline vs Wk5 biopsies showed decreased ER, PR, and Ki67 levels by Wk5 that remained decreased at time of surgery. AR and GR were significantly decreased only in the FE arm at the time of surgery. RPPA revealed that baseline EGFR (Y1604 and Y992) was higher in tumors with PEPI=0 tumors, whereas the average baseline mTOR activation was higher among pts with PEPI>0. Significant changes detected by RPPA upon treatment included a significant decrease in AR. Myeloid-derived suppressor cells (MDSC) were significantly reduced by treatment only in the FE arm. Conclusions: The combination FE had manageable side effects. PEPI=0 was achieved more frequently on the FE arm (8/33) than the F arm (2/26). Activated EGFR was higher pretreatment in tumors achieving PEPI=0. mTOR pathway was elevated pretreatment in PEPI>0 tumors (also observed with resistance to FE in ER+/HER2- metastatic disease (SABCS 2021). When comparing pretreatment tumor to Wk5, total AR by RPPA was the most differentially decreased protein in PEPI=0 tumors. AR signaling is known to support immunosuppressive cells and we observed a marked decrease in MDSCs with treatment only on the FE arm. Citation Format: Anthony D. Elias, Alyse Staley, Monica Fornier, Gregory A. Vidal, Sharon Sams, Nicole Spoelstra, Peter Kabos, Jennifer R. Diamond, Elena Shagisultanova, Rosa I. Gallagher, Julia Wulfkuhle, Emanuel Petricoin, Kathryn Zolman, Stephanie Biller, Vida Alami, Tessa McSpadden, Virginia Borges, Lyndsey S. Crump, Dexiang Gao, Jennifer K. Richer. Randomized phase II trial of preoperative fulvestrant with or without enzalutamide for ER+/Her2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT144.

Abstract 6674: Single-dose trilaciclib monotherapy potentiates antitumor immunity in the neoadjuvant setting of triple-negative breast cancer by modulating the composition and effector function of peripheral immune cells

Abstract Intravenous administration of trilaciclib transiently arrests cyclin-dependent kinase 4/6-dependent cells in the G1 phase of the cell cycle during chemotherapy exposure. In an open-label, phase 2 study in patients with metastatic triple-negative breast cancer (TNBC; NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin improved overall survival and resulted in enriched T-cell diversity and decreased clonality in peripheral blood compared with administering chemotherapy alone. This phase 2, single-arm, neoadjuvant study (NCT05112536) aims to determine the immune mechanism of action of trilaciclib and its role in modulating antitumor immune responses in patients with TNBC. Previously untreated patients with early-stage TNBC (N = 24) received single-dose trilaciclib monotherapy during the lead-in phase. After 7 days, patients received systemic therapy with 4 cycles of dose-dense doxorubicin plus cyclophosphamide and 12 weekly cycles of paclitaxel, with trilaciclib administered prior to the first dose of systemic therapy per cycle. Pembrolizumab from cycle 1 and/or carboplatin from cycle 5 was permitted per investigator discretion. Patients underwent curative surgery 3-5 weeks after the last dose of systemic therapy. Peripheral blood samples were collected at baseline, 7 (±1) days post trilaciclib monotherapy prior to chemotherapy, and pre dose on cycle 2, day 1. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and cryopreserved for immune profiling via cytometry by time-of-flight (CyTOF), and for functional analysis via intracellular cytokine staining (ICS) and dimensionality reduction. A preliminary analysis of PBMCs from 7 patients showed a decrease in peripheral myeloid-derived suppressor cells (MDSCs), monocytes, and classical dendritic cells following single-dose trilaciclib monotherapy. There were nominal changes to the frequency of peripheral CD4+ and CD8+ T cells. In 4/7 patients, trilaciclib monotherapy resulted in increased proportions of activated and polyfunctional CD4+ and CD8+ T cells producing IFNγ, TNFα, and IL-2 cytokines. Furthermore, trilaciclib enhanced the frequency of CD8+ T cells producing granzyme B and perforin. The decrease in MDSCs and increase in functional T cells following trilaciclib monotherapy supports findings from an immune analysis of data from the phase 2 study of trilaciclib in patients with metastatic TNBC. Overall, these data suggest that the transient G1 arrest of peripheral immune cells by single-dose trilaciclib monotherapy favorably modulates immune responses by promoting the generation of polyfunctional T cells and limiting the number of MDSCs in circulation. Enrollment was completed in August 2022. The complete dataset from all 24 patients will be presented. Citation Format: Sarah Ahn, Subing Cao, Amanda Jacobson, Melissa A. Russo, John S. Yi. Single-dose trilaciclib monotherapy potentiates antitumor immunity in the neoadjuvant setting of triple-negative breast cancer by modulating the composition and effector function of peripheral immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6674.

Abstract 4503: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplasia which contributes to poor prognosis by hindering tumor entry of immune cells and therapeutics. This leads to PDAC being difficult to treat with gemcitabine, a current standard-of-care, with only a 22% 12-month survival rate. Therapeutic modalities are needed to tackle the PDAC tumor microenvironment (TME). An alternative target is polyamine metabolism which is required for tumor growth, survival, metastasis, and also for modulating generation, survival, and activity of immune cells. Myeloid-derived suppressor cells (MDSCs) are immune cells that have a key role in PDAC metastasis, invasion, progression, and in impeding anti-tumor immune response to therapeutics in PDAC. MDSCs include monocyte-like (M-MDSC) and neutrophil-like (PMN-MDSC) cells. Previous studies have shown that polyamine blockade therapy (PBT), consisting of difluoromethylornithine (DFMO) to block polyamine biosynthesis and a polyamine transport inhibitor (Trimer44NMe), elicits anti-tumor immune response, improved survival, and a decrease in MDSCs in other tumor types. This is of interest given our previous findings that PBT improved survival of PDAC-bearing mice. Here, we tested PBT in altering PDAC-stimulated MDSC survival and whether it shifts their immunosuppressive phenotype. Studies were conducted using CD11b+ bone marrow cells that were differentiated into MDSCs via GM-CSF and tumor-conditioned media from PDAC cells derived from a genetic mouse model for pancreatic cancer (KPC-mice). Bulk RNA-sequencing was conducted on MDSCs treated with PBT, gemcitabine or combination. MDSCs exhibited widespread downregulation of expression in the gemcitabine and combination treatment, but PBT alone exhibited more targeted downregulation. Specifically, Gene Ontology (GO) analysis revealed a decrease in genes associated with immune cell migration and chemotaxis, and KEGG analysis showed downregulation of inflammatory pathways. These findings indicate that PBT may modify MDSCs from their tumor-associated immunosuppressive and pro-inflammatory behavior. MDSC subtype-specific markers were investigated by flow cytometry. Results show subsets of immune cells, such as PMN-MDSCs, are reduced upon PBT treatment alone or in combination with gemcitabine, thereby highlighting the dependence of this subtype on polyamine metabolism. In conclusion, PBT alters the expression of MDSC immunomodulatory genes, decreases MDSC survival and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode, Sai Preethi Nakkina, Manav Gandhi, Otto Phanstiel, Deborah A. Altomare. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4503.

Biomimetic Gold Nanostructure with a Virus-like Topological Surface for Enhanced Antigen Cross-Presentation and Antitumor Immune Response.

The internalization of antigens by dendritic cells (DCs) is the initial critical step for vaccines to activate the immune response; however, the systemic delivery of antigens into DCs is hampered by various technical challenges. Here we show that a virus-like gold nanostructure (AuNV) can effectively bind to and be internalized by DCs due to its biomimetic topological morphology, thereby significantly promoting the maturation of DCs and the cross-presentation of the model antigen ovalbumin (OVA). In vivo experiments demonstrate that AuNV efficiently delivers OVA to draining lymph nodes and significantly inhibits the growth of MC38-OVA tumors, generating a ∼80% decrease in tumor volume. Mechanistic studies reveal that the AuNV-OVA vaccine induces a remarkable increase in the rate of maturation of DCs, OVA presentation, and CD4+ and CD8+ T lymphocyte populations in both lymph node and tumor and an obvious decrease in myeloid-derived suppressor cells and regulatory T cell populations in spleen. The good biocompatibility, strong adjuvant activity, enhanced uptake of DCs, and improved T cell activation make AuNV a promising antigen delivery platform for vaccine development.

In Situ Reprogramming of Tumor-Associated Macrophages with Internally and Externally Engineered Exosomes.

The reprogramming of tumor-associated macrophages (TAMs) has emerged as an efficient strategy for immunotherapy. However, most of the approaches did not allow the in situ reprogramming of TAM because their low efficiency, non-specificity, or potential side effects. Herein, we produced exosomes with the clustered regularly interspaced short palindromic repeats interference (CRISPRi) internally engineered and the TAM specific peptide externally engineered onto the exosome membrane. The internally and externally engineered exosomes (IEEE, also named as I3E) allowed the selective homing to tumor tissue and targeted to M2-like TAMs, which nearly repressed the expression of PI-3 kinase gamma (PI3Kγ) completely, and induced the TAMs polarizing to M1 both in vitro and in vivo. The polarized M1 macrophages awakened the "hot" tumor-immunity, causing the increase of T lymphocyte infiltration and the decrease of myeloid-derived suppressor cells, and inhibiting the tumor growth significantly. I3E reprogramed TAMs in situ precisely and efficiently.

Blockade of EP4 by ASP7657 Modulates Myeloid Cell Differentiation In Vivo and Enhances the Antitumor Effect of Radiotherapy.

The tumor microenvironment (TME) is thought to influence the antitumor efficacy of immuno-oncology agents through various products of both tumor and stromal cells. One immune-suppressive factor is prostaglandin E2 (PGE2), a lipid mediator whose biosynthesis is regulated by ubiquitously expressed cyclooxygenase- (COX-) 1 and inducible COX-2. By activating its receptors, PGE2 induces immune suppression to modulate differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) rather than dendritic cells (DCs). Pharmacological blockade of prostaglandin E receptor 4 (EP4) causes a decrease in MDSCs, reprogramming of macrophage polarization, and increase in tumor-infiltrated T cells, leading to enhancement of antitumor immunity in preclinical models. Here, we report the effects of the highly potent EP4 antagonist ASP7657 on the DC population in tumor and antitumor immune activation in an immunocompetent mouse tumor model. Oral administration of ASP7657 inhibited tumor growth, which was accompanied by an increase in intratumor DC and CD8+ T cell populations and a decrease in the M-MDSC population in a CT26 immunocompetent mouse model. The antitumor activity of ASP7657 was dependent on CD8+ T cells and enhanced when combined with an antiprogrammed cell death-1 (PD-1) antibody. Notably, ASP7657 also significantly enhanced the antitumor efficacy of radiotherapy in an anti-PD-1 antibody refractory model. These results indicate that the therapeutic potential of ASP7657 arises via upregulation of DCs and subsequent CD8+ T cell activation in addition to suppression of MDSCs in mouse models and that combining EP4 antagonists with radiotherapy or an anti-PD-1 antibody can improve antitumor efficacy.

Inhibition of Orthotopic Genital Cancer Induced by Subcutaneous Administration of Human Papillomavirus Peptide Vaccine with CpG Oligodeoxynucleotides as an Adjuvant in Mice.

PurposePersistent high-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer and its precursor lesions. Although prophylactic HPV vaccines have been applied in the general population for the prevention of HPV infections, no licensed therapeutic HPV vaccine is currently available to treat preexisting HPV infections or HPV-associated diseases, including cervical cancer.Materials and MethodsThe most common murine cervical cancer model used for the evaluation of the efficacy of a therapeutic HPV vaccine in preclinical studies is the ectopic model, which is established by the subcutaneous inoculation of tumor cells, such as TC-1 cells, into the flank of an animal. We have previously demonstrated the efficacy of a therapeutic HPV peptide vaccine adjuvanted with unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide in the clearance of ectopic subcutaneous tumors in C57BL/6 mice after vaccination. In the current study, we established orthotopic genital tumors by injecting TC-1 cells into the vaginal submucosa close to the cervix and assessed whether the subcutaneous administration of the therapeutic vaccine could inhibit the growth of genital tumors. Additionally, we evaluated the effect of the vaccination on the tumor microenvironment.ResultsThe results showed that the vaccination induced an increase in infiltrating CD4+ and CD8+ T cells, a decrease in myeloid-derived suppressor cells and cancer-associated fibroblasts, as well as the differential expression of a panel of cytokines, chemokines, and matrix metalloproteinases within the tumor microenvironment.ConclusionThe administration of the vaccine resulted in the inhibition of established implanted orthotopic genital tumors by inducing strong antitumor immune responses and reversed tolerogenic local immunosuppression in a mouse model of orthotopic genital cancer.

Increased Regulatory T Cells and Decreased Myeloid-Derived Suppressor Cells Induced by High CCL17 Levels May Account for Normal Incidence of Cancers among Patients with Atopic Dermatitis.

The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-dominant environment is known to downregulate tumor immunity. To gain mechanistic insights regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency of myeloid-derived suppressor cells (MDSCs), however, was significantly decreased in tumors of CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17 attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a “safety-net” to reduce the risk of malignant tumors in the Th2-dominant environment.

Abstract 3423: CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy

Abstract Recent literature demonstrates that tumor hijacks physiological mechanisms of C-type lectin receptors (CLRs) that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promotes immune escape. We previously demonstrated that the orphan CLR CLEC-1 expressed by myeloid cells, is enhanced by the immunosuppressive cytokine TGFβ and tempers downstream CD4+ Th1 and Th17 responses following sterile inflammation. Interestingly, we observed a high CLEC-1 expression by human CD11b+ myeloid cells from ovarian ascites and from in-vitro generated pro-tumoral M2 macrophages. Moreover, open-source data show that Clec1a is highly expressed by XCR1-expressing mouse dendritic cells (DC). As these cDC1 are specialized in cross-presentation of dead cell-associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells and regulate anti-tumor immunity. Interestingly, we observed that both human and mouse CLEC-1 bind specifically to secondary necrotic cells. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that the ligand(s) of CLEC-1 correspond(s) to Damage-Associated Molecular Pattern(s) induced by the cell death process. Then, to test if CLEC-1 in DCs influences cross-presentation of dead cell-associated antigens, we immunized WT and Clec1a KO mice with OVA-loaded dead cells and co-injected OVA-specific TCR transgenic CD8+ OT-I T cells. Impressively, we observed an increased proliferation of CD8+ T cells in the absence of CLEC-1, demonstrating that CLEC-1 in DCs tempers cross-presentation of dead cell-associated antigens. Interestingly, we observed in a model of subcutaneous MC38 colon carcinoma that the combination of the absence of CLEC-1 with cytotoxic and immunogenic chemotherapy (cyclophosphamide), reduces significantly the tumor growth and cures most of the mice. Similarly, in orthotopic models of mesothelioma (AK7) or hepatocarcinoma (Hepa1.6), we observed a prolongation of survival of Clec1a KO mice. Mechanistically, this was associated with an increase of effector CD69+ and of central memory CD44hi CD62Lhi CCR7hi CD4+ and CD8+ T cells in both liver and spleen. Furthermore, we observed a decrease in myeloid-derived suppressor cells. Besides, macrophages display a more mature phenotype relative to MHC class II expression. In addition, transcriptomics analysis performed in tumor-bearing liver of Clec1a KO mice revealed a profound decrease in CSF1 expression suggesting a defect in immunosuppressive myeloid cell recruitment. Moreover, we observed an increase in CCL17 and CCL7 expression, both known to activate CD8+ T cells through DCs and to enhance anti-tumor immunity. Taken together, these results suggest that CLEC-1 in DCs by acting as sensor of cell damage tempers immune response and represents a novel pharmacological target for cancer immunotherapy. Citation Format: Marion Drouin, Javier Saenz, Bérangère Evrard, Vanessa Gauttier, Géraldine Teppaz, Maria-Dolores Lopez-Robles, Cédric Louvet, Nicolas Poirier, Elise Chiffoleau. CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3423.

Decreased Circulating Myeloid Derived Suppressor Cells at First Follow-up Predict Favorable Response to Immunotherapy in a Randomized Trial of Nivolumab and Bevacizumab in Recurrent GBM

Abstract INTRODUCTION A potential barrier to successful immunotherapy response in glioblastoma (GBM) is myeloid-derived suppressor cells (MDSCs): an immature immunosuppressive cell elevated in the circulation and tumor of GBM patients. Given a limited set of biomarkers predictive of response to immunotherapy in GBM, we explored the change in MDSC levels with immunotherapy to predict treatment response. METHODS A retrospective analysis was performed on patients in a randomized, phase 2 study of nivolumab and bevacizumab at GBM first recurrence. Clinical and radiographic data were analyzed for disease progression or treatment response via Response Assessment in Neuro-Oncology (RANO) criteria. Blood was collected prior to treatment and at first imaging follow-up. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and analyzed. Characterization of circulating immune cells was performed using flow cytometry. RESULTS A total of 9 patients were identified as responders or nonresponders at a median time of 8.7 wk (range 6.9-10.0) after therapy initiation. MDSCs, as a percentage of total PBMCs, were elevated at baseline in responders compared to nonresponders (4.9% ± 0.7 vs 2.6% ± 0.2, P = .019), which reversed at follow up (1.8% ± 0.4 vs 5.8% ± 1.1, P = .032). There was a 6.4 fold decrease in MDSCs as a percentage of total PBMCs between baseline and first imaging follow-up in responders as compared to nonresponders (P = .001). When looking at subtypes of MDSCs, a 4.9 fold decrease in granulocytic MDSCs (G-MDSCs) was noted in responders over nonresponders (P = .010). Further investigation of this cohort by simultaneous single-cell analysis of transcriptome and surface epitopes is ongoing. CONCLUSION Differences in circulating MDSC levels that were specific to responders and nonresponders were seen both before and after therapy initialization, with decreases in MDSCs (specifically G-MDSCs) being correlated with treatment response. Characterization of MDSCs in the peripheral blood may be helpful in identifying GBM patients likely to benefit from immunotherapy.

Abstract 3969: TP-0903, a potent AXL receptor tyrosine kinase inhibitor, enhances the activity of anti-PD-1 therapy in a metastatic preclinical syngeneic model of breast cancer

Abstract The high rate of non-responders to immune checkpoint inhibitors (ICI) represents significant challenges in the field of immune-oncology. Breast cancer has emerged as a cancer type that responds poorly to ICI. Therefore, there is an urgent need to better understand how cancer cells escape immune surveillance and resist immune attacks. Cancer cell clones with mesenchymal features seem to be less susceptible to attacks by immune cells. Hence, epithelial-to-mesenchymal transition (EMT) targeting agents such as TP-0903 are emerging as potential combination candidates to enhance immune checkpoint inhibition. AXL is overexpressed in a variety of cancers, often driving the pathogenesis and progression of disease. AXL overexpression induces cancer growth, invasion, metastasis, drug resistance, and mesenchymal characteristics. Aside from cancer cells, AXL is also expressed on several types of immune cells including macrophages and dendritic cells. Recently, it has been reported that AXL plays an important role as a negative regulator of immune response, contributing to an anti-tumor immune suppression. We have previously identified TP-0903 to be a potent small molecule inhibitor of AXL (IC50=14 nM) capable of reversing EMT. In this study, we evaluated whether TP-0903 could enhance the ICB effect in an ICB-resistant triple negative, metastatic breast cancer mouse model (4T1). In the 4T1 syngeneic model, anti-PD-1 monotherapy failed to inhibit tumor growth indicating that this tumor is resistant to anti-PD-1 treatment. The combination of TP-0903 and anti-PD-1 resulted in statistically significant tumor growth inhibition versus TP-0903 monotherapy (p<0.05). We hypothesized that the combination effect was influenced by potential CD8+ T cell depletion, and further investigated the effects of TP-0903 on immune cells in spleen and tumors. Tumor growth inhibition was found to be associated with a decrease in myeloid-derived suppressor cells (MDSC) in the spleen and an increase in infiltration and activation of dendritic cells (DCs) in the tumor. Gene expression analysis revealed that TP-0903 treatment decreased multiple immunosuppressive cytokines and chemokines including IL-6 and G-CSF in vivo. These results suggest that TP-0903 modulates the immune-suppressive tumor microenvironment (TME) to reinvigorate T cell immunity in anti-PD-1 resistant 4T1 tumors. In conclusion, AXL inhibition with TP-0903 modulates TME and enhances the anti-tumor effects of ICBs in an anti-PD-1 resistant, metastatic breast cancer mouse model. These findings support additional clinical testing of TP-0903 in combination with ICBs, which is currently being investigated in patients with advanced solid tumors in an ongoing clinical study (NCT02729298). Citation Format: Yuka Kumagai, Jun Oishi, Megumi Nakamura, Jason M. Foulks, Clifford J. Whatcott, Steven L. Warner, David J. Bearss, Masashi Goto. TP-0903, a potent AXL receptor tyrosine kinase inhibitor, enhances the activity of anti-PD-1 therapy in a metastatic preclinical syngeneic model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3969.

Inhibiting the CD8+ T cell infiltration in the tumor microenvironment after radiotherapy is an important mechanism of radioresistance

Endogenous immune response participates in tumor control, and radiotherapy has immune modulatory capacity, but the role of immune modulation in the tumor microenvironment invoked by radiotherapy in radiosensitivity is poorly defined. In the present study, a radio-resistant melanoma cell line was obtained after repeated irradiation to the parental tumor in C57BL/6 mice. Radiotherapy resulted in aggregation of CD8+ and CD3+ T cells, and decrease of myeloid-derived suppressor cells and dendritic cells in the parental tumor, but not in the resistant tumors. CD4+ T cells and B cells did not change significantly. The CD8+ T cell infiltration after radiotherapy is important for tumor response, because in the nude mice and CD8+ T cell-depleted C57BL/6 mice, the parental and resistant tumor has similar radiosensitivity. Patients with good radiation response had more CD8+ T cells aggregation after radiotherapy. Radiotherapy resulted in robust transcription of T cell chemoattractant in the parental cells, and the expression of CCL5 was much higher. These results reveal a novel mechanism of radioresistance, tumor cells inhibit the infiltration of CD8+ T cell after radiotherapy and become radioresistant. Increasing CD8+ T cell infiltration after RT may be an effective way to improve tumor radiosensitivity.

Abstract 2753: Translation to the clinic of EVT801: A novel immune-oncology agent for addressing innate-driven immunosuppression into the tumor microenvironment and expanding patient population responding to immune checkpoint therapies

Abstract Amongst resistance mechanisms of immune checkpoint therapies (ICTs), expansion and recruitment of immunosuppressive innate cells and in particular Myeloid Derived Suppressor Cells (MDSCs) can be a major cause of resistance to ICTs. Here we describe joint efforts of clinicians and researchers to translate the promising EVT801 action on MDSCs into the clinic. We have identified a highly selective drug candidate targeting VEGFR3, EVT801, showing intermediate in vivo activity on tumours, accompanied by a decrease of MDSCs, when VEGFR3 is expressed in the tumour microenvironment. To evaluate the potential to combine with ICTs, the 4T1 orthotopic mammary carcinoma mouse model, was used. We have demonstrated that EVT801 increases the therapeutic activity of an anti-PD-1 antibody (Ab) on primary tumours as compared to single agent treatment but also results in a decrease of lung metastasis. Furthermore, the decrease of MDSCs in the blood in response to EVT801 is correlated with tumor shrinkage and therapeutic efficacy. In parallel, Immuno Histo Chemistry analyses have shown that treatment with EVT801 increases CD8+ T-cells infiltration inside the tumor. Taken together, these results indicate that EVT801 represents an innovative drug for cancer immunotherapy that improves the frequency of response to ICT by controlling MDSCs immunosuppressive activity and in turn by unleashing tumor-specific T-lymphocytes. To translate these promising results into the clinic, we have evaluated different biomarkers to properly select patients and monitor efficacy of EVT801: (1) We refined an antiPD1 Ab resistance gene signature and correlated it with VEGFR3 pathway gene overexpression. (2) We investigated VEGFR3 expression in the tumor microenvironment (TME) for different cancer indications. Taken together, these results will facilitate patient stratification as based on the PD1 resistance gene signature associated with VEGFR3 expression in the TME. (3) In non-small cell lung cancer patients, we validated that a high-level baseline of circulating M-MDSCs is associated with poor survival. In correlation with EVT801 activity on circulating MDSC as deciphered on preclinical mouse models, we propose that circulating MDSC levels could be a biomarker of activity for EVT801 in non-responder patients to ICTs. EVT801 represents a novel agent for cancer immunotherapy for non-responder patients to ICT. Patient stratification strategy, target engagement biomarker and biomarkers of activity have been identified and will enable the initiation of a planned Phase I clinical trial with EVT801. Citation Format: Pierre Fons, Michael Esquerre, Julien Mazieres, Philippe Rochaix, Anne Gomez-Brouchet, Antoine Alam, Florie Bertrand, Celine Poussereau-Pomie, Jerome Meneyrol, Anne Pradines, janick selves, Isabelle Rouquette, Isabelle Blanc, Francoise Bono, Donogh P. O'Brien, Michael Paillasse, Joanna Lisztwan, Mark Whittaker. Translation to the clinic of EVT801: A novel immune-oncology agent for addressing innate-driven immunosuppression into the tumor microenvironment and expanding patient population responding to immune checkpoint therapies [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 2753.

Snail promotes ovarian cancer progression by recruiting myeloid-derived suppressor cells via CXCR2 ligand upregulation

Snail is a major transcriptional factor that induces epithelial-mesenchymal transition (EMT). In this study, we explore the effect of Snail on tumor immunity. Snail knockdown in mouse ovarian cancer cells suppresses tumor growth in immunocompetent mice, associated with an increase of CD8+ tumor-infiltrating lymphocytes and a decrease of myeloid-derived suppressor cells (MDSCs). Snail knockdown reduces the expression of CXCR2 ligands (CXCL1 and CXCL2), chemokines that attract MDSCs to the tumor via CXCR2. Snail upregulates CXCR ligands through NF-kB pathway, and most likely, through direct binding to the promoters. A CXCR2 antagonist suppresses MDSC infiltration and delays tumor growth in Snail-expressing mouse tumors. Ovarian cancer patients show elevated serum CXCL1/2, which correlates with Snail expression, MDSC infiltration, and short overall survival. Thus, Snail induces cancer progression via upregulation of CXCR2 ligands and recruitment of MDSCs. Blocking CXCR2 represents an immunological therapeutic approach to inhibit progression of Snail-high tumors undergoing EMT.