CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on ...antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8(+) T cells and associated with an increased frequency of antigen-specific CD8(+) T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)-depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells.
It has been suggested that marrow stromal cells (MSCs) may be immunoprivileged and can engraft in allogeneic recipients with intact immune systems. We determined if the implantation of murine MSCs ...engineered to release erythropoietin (Epo) would be feasible in major histocompatibility complex (MHC)-mismatched allogeneic mice without immunosuppression, and we monitored hematocrit (Hct) as a reporter of MSC graft survival. MSCs from C57Bl/6 mice were engineered to release murine Epo (Epo+ MSCs) and implanted subcutaneously in either syngeneic C57Bl/6 mice or MHC-mismatched Balb/c mice. In syngeneic recipients, the Hct rapidly rose from baseline level and remained higher than .88 (88%) for more than 200 days. However, in MHC-mismatched recipient Balb/c mice, the Hct rose transiently and rapidly declined to baseline values. Repeat implantations in these same mice were associated with an acquired refractoriness in the Hct response consistent with alloimmunization to donor Epo+ MSCs. Allogeneic MSC implants had an increased proportion of host-derived lymphoid CD8+, natural killer T (NKT), and NK infiltrating cells compared with syngeneic controls, and splenocytes isolated from Balb/c mice that had received implants also displayed a significant interferon-gamma (IFNγ) response to C57Bl/6 MSCs in vitro. These results strongly suggest that MSCs are not intrinsically immunoprivileged and cannot serve as a “universal donor” in immunocompetent MHC-mismatched recipients.
CD73 is a cell surface 5'-nucleotidase that converts AMP to adenosine, an immune suppressive molecule. CD73 may promote immune escape in cancer by contributing to the degradation of extracellular ATP ...released by dying cancer cells in hypoxic tumors or following chemotherapy. However, whether CD73 exerts a critical oncogenic function during tumorigenesis is unknown. In this study, we used genetically deficient mice to investigate its contribution to autochthonous tumor formation. CD73 deficiency suppressed the development of 3-methylcholanthrene (MCA)-induced fibrosarcomas through a mechanism relying upon IFN-γ, natural killer (NK) cells, and CD8(+) T cells. Similarly, CD73 deficiency also suppressed prostate tumorigenesis in TRAMP transgenic mice. Importantly, treatment with an anti-CD73 monoclonal antibody effectively suppressed growth of established MCA-induced tumors or TRAMP-C1 prostate tumors and inhibited the development of TRAMP-C1 lung metastases. The therapeutic activity of anti-CD73 monoclonal antibody against primary tumors was dependent on CD8(+) T cells, whereas its antimetastatic activity was dependent on host CD73 expression independent of T cells or NK cells. Taken together, our findings indicate that CD73 is a critical factor in tumorigenesis and that anti-CD73 antibodies may offer a novel generalized strategy to blunt immune escape and treat cancer.
Abstract BACKGROUND: Therapeutic strategies targeting immune checkpoint proteins have led to significant responses in patients with various tumor types. The success of these studies has led to the ...development of various antibodies/inhibitors for the different checkpoint proteins involved in immune evasion of the tumor. Adenosine present in high concentrations in the tumor microenvironment activates the immune checkpoint adenosine A2a receptor (A2aR), leading to the suppression of antitumor responses. Inhibition of this checkpoint has the potential to enhance antitumor T-cell responsiveness. METHODS: We developed a novel A2aR antagonist (PBF-509) and tested its antitumor response in vitro , in a mouse model, and in non-small cell lung cancer patient samples. RESULTS: Our studies showed that PBF-509 is highly specific to the A2aR as well as inhibitory of A2aR function in an in vitro model. In a mouse model, we found that lung metastasis was decreased after treatment with PBF-509 compared with its control. Furthermore, freshly resected tumor-infiltrating lymphocytes from lung cancer patients showed increased A2aR expression in CD4+ cells and variable expression in CD8+ cells. Ex vivo studies showed an increased responsiveness of human tumor-infiltrating lymphocytes when PBF-509 was combined with anti-PD-1 or anti-PD-L1. CONCLUSIONS: Our studies demonstrate that inhibition of the A2aR using the novel inhibitor PBF-509 could lead to novel immunotherapeutic strategies in non-small cell lung cancer.
The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the ...relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4
+
T cells and CD19
+
B cells and a lower mean frequency of CD8
+
T cells compare with normal tissues, increasing the CD4
+
/CD8
+
T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4
+
T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.
The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process ...is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro. Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors.
In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.
The efficacy of immunotherapy targeting the PD-1/PD-L1 pathway has previously been demonstrated in metastatic head and neck squamous cell carcinoma (HNSCC). Stereotactic Body Radiotherapy (SBRT) aims ...at ablating metastatic lesions and may play a synergistic role with immunotherapy. The purpose of this study is to assess the safety and efficacy of triple treatment combination (TTC) consisting of the administration of durvalumab and tremelimumab in combination with SBRT in metastatic HNSCC.
This is a phase I/II single arm study that will include 35 patients with 2-10 extracranial metastatic lesions. Patients will receive durvalumab (1500 mg IV every 4 weeks (Q4W)) and tremelimumab (75 mg IV Q4W for a total of 4 doses) until progression, unacceptable toxicity or patient withdrawal. SBRT to 2-5 metastases will be administered between cycles 2 and 3 of immunotherapy. The safety of the treatment combination will be evaluated through assessment of TTC-related toxicities, defined as grade 3-5 toxicities based on Common Terminology Criteria for Adverse Events (v 4.03), occurring within 6 weeks from SBRT start, and that are definitely, probably or possibly related to the combination of all treatments. We hypothesize that dual targeting of PD-L1 and CTLA-4 pathways combined with SBRT will lead to < 35% grade 3-5 acute toxicities related to TTC. Progression free survival (PFS) will be the primary endpoint of the phase II portion of this study and will be assessed with radiological exams every 8 weeks using the RECIST version 1.1 criteria.
The combination of synergistic dual checkpoints inhibition along with ablative radiation may significantly potentiate the local and systemic disease control. This study constitutes the first clinical trial combining effects of SBRT with dual checkpoint blockade with durvalumab and tremelimumab in the treatment of metastatic HNSCC. If positive, this study would lead to a phase III trial testing this treatment combination against standard of care in metastatic HNSCC.
NCT03283605 . Registration date: September 14, 2017; version 1.
Many individuals at risk of streptococcal infection respond poorly to the pneumococcal polysaccharide vaccine Pneumovax 23. Identification of actionable pathways able to enhance Pneumovax ...responsiveness is highly relevant. We investigated the contribution of the extracellular adenosine pathway regulated by the ecto-nucleotidase CD73 in Pneumovax-induced antibody responses. Using gene-targeted mice, we demonstrated that CD73-or A2a adenosine receptor deficiency significantly delayed isotype switching. Nevertheless, CD73- or A2aR- deficient adult mice ultimately produced antigen-specific IgG3 and controlled Streptococcus pneumoniae infection as efficiently as wild type (WT) mice. Compared to adults, young WT mice failed to control S. pneumoniae infection after vaccination and this was associated with lower levels of CD73 on innate B cells. We hypothesized that pharmacological activation of A2a receptor may improve Pneumovax 23 immunization in young WT mice. Remarkably, administration of the A2a adenosine receptor agonist CGS 21680 significantly increased IgG3 responses and significantly enhanced survival after S. pneumoniae challenge. Our study thus suggests that pharmacological activation of the A2a adenosine receptor could improve the efficacy of Pneumovax 23 vaccination in individuals at risk of streptococcal infection.
CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large ...randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.
Finding a noninvasive radiomic surrogate of tumor immune features could help identify patients more likely to respond to novel immune checkpoint inhibitors. Particularly, CD73 is an ectonucleotidase ...that catalyzes the breakdown of extracellular AMP into immunosuppressive adenosine, which can be blocked by therapeutic antibodies. High CD73 expression in colorectal cancer liver metastasis (CRLM) resected with curative intent is associated with early recurrence and shorter patient survival. The aim of this study was hence to evaluate whether machine learning analysis of preoperative liver CT-scan could estimate high vs low CD73 expression in CRLM and whether such radiomic score would have a prognostic significance.
We trained an Attentive Interpretable Tabular Learning (TabNet) model to predict, from preoperative CT images, stratified expression levels of CD73 (CD73
vs. CD73
) assessed by immunofluorescence (IF) on tissue microarrays. Radiomic features were extracted from 160 segmented CRLM of 122 patients with matched IF data, preprocessed and used to train the predictive model. We applied a five-fold cross-validation and validated the performance on a hold-out test set.
TabNet provided areas under the receiver operating characteristic curve of 0.95 (95% CI 0.87 to 1.0) and 0.79 (0.65 to 0.92) on the training and hold-out test sets respectively, and outperformed other machine learning models. The TabNet-derived score, termed rad-CD73, was positively correlated with CD73 histological expression in matched CRLM (Spearman's ρ = 0.6004; P < 0.0001). The median time to recurrence (TTR) and disease-specific survival (DSS) after CRLM resection in rad-CD73
vs rad-CD73
patients was 13.0 vs 23.6 months (P = 0.0098) and 53.4 vs 126.0 months (P = 0.0222), respectively. The prognostic value of rad-CD73 was independent of the standard clinical risk score, for both TTR (HR = 2.11, 95% CI 1.30 to 3.45, P < 0.005) and DSS (HR = 1.88, 95% CI 1.11 to 3.18, P = 0.020).
Our findings reveal promising results for non-invasive CT-scan-based prediction of CD73 expression in CRLM and warrant further validation as to whether rad-CD73 could assist oncologists as a biomarker of prognosis and response to immunotherapies targeting the adenosine pathway.
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