CD47 Inhibition Research Articles

CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis.

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2+ breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

Abstract NT-107: CHEMORESISTANT OVARIAN CANCER STEM CELLS REVEAL NOVEL THERAPEUTIC TARGETS

Abstract More than 60% of patients with ovarian cancer respond to first-line therapy, often achieving complete remission. However, these encouraging clinical outcomes are quickly overshadowed by high rates of treatment failure. More than half of advanced-stage patients relapse within five years, and these patients face poor prognoses. We sought to better understand the cause of ovarian cancer treatment failure with the hope of improving patient outcomes. We hypothesized that ovarian cancer stem cells may resist chemotherapy allowing them to regrow tumors causing relapse, metastasis and ultimately death. We further postulated that characterization of these cells would reveal therapeutic targets that may be exploited to achieve more durable remissions. To begin to test this hypothesis, we proposed that high aldehyde dehydrogenase I activity (ALDH1high) would identify stem-like ovarian cancer cells that would resist chemotherapy treatment and be capable of regenerating tumors. We compared ALDH1high cells to ALDH1low cells in three ovarian cancer cell lines: the human SKOV3 cell line, the rat FNAR-C1 cell line, and the murine ID8 cell line. Consistent with our predictions, we found that ALDH1high cells were resistant to several drugs commonly used to treat ovarian cancer. The ALDH1high population was also the only fraction of cells capable of forming tumors in vivo. In keeping with their proposed cancer stem cell phenotype, ALDH1high cells were smaller in size, relatively quiescent, and able to regenerate the phenotypic diversity of the cell line. Furthermore, they lacked contact inhibition and were capable of nonadherent growth. Significant controversy exists concerning how best to identify ovarian cancer stem cells. Many markers have been proposed with data to both support and refute their use in the literature. Resolving these uncertainties is hampered by the lack of systematic evaluation of each marker in proposed cancer stem cell pools. We used microarray analysis and quantitative real-time RT-PCR to measure the expression of ABCG2, CD24, CD44, CD133 and KIT in our ALDH1high cells compared to their ALDH1low counterparts and found no consistent differential regulation in the ALDH1high cells across all three cell lines. Further work remains needed to more clearly define the relationships between each of these proposed ovarian cancer stem cell markers. Gene expression profiles identified several possible vulnerabilities that could be exploited therapeutically. ALDH1high cells have upregulation of the mTOR pathway, FGF18 and CD47. They also express Her-2/neu at similarly high levels as ALDH1low cells. Drugs are already clinically available to target mTOR and Her-2/neu, which would speed their implementation. FGF18 signals through the FGFR3 receptor. Although FGF18 is upregulated in the cancer stem cell pool, both cancer stem and non-stem cells express its receptor, FGFR3, at similar levels. Several approaches to target FGFR3 are in clinical trials, as are antibodies targeting CD47. mTOR and CD47 inhibition would potentially be selective for ovarian cancer stem cells, but Her-2/neu and FGFR3 inhibition could also target the more differentiated, non-stem cancer cells. Further work is necessary to validate these treatment approaches. Citation Format: Allison C. Sharrow, Richard J Jones, Lily Wu. CHEMORESISTANT OVARIAN CANCER STEM CELLS REVEAL NOVEL THERAPEUTIC TARGETS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-107.

Inhibition of CD47 as a Novel Cancer Immunotherapy for Multiple Myeloma

CD47 is a protein highly expressed on the surface of various cancer cells, allowing them to escape innate immune recognition by sending inhibitory signals to macrophages (Ma), which further hinders the anti-tumor response by the adaptive immune system. This CD47

Combined prognostic value of the cancer stem cell markers CD47 and CD133 in esophageal squamous cell carcinoma.

BackgroundTreatments based on the inhibition of pivotal signals of cancer stem cells (CSCs) are on a promising track. Recent studies have shown that targeting CSCs with broader immune‐based therapeutic methods, for example, the anti‐CD47 treatment, may serve as a more potent strategy for eliminating these intractable cells. We aimed to explore the prognostic effects of CD47/CD133 and the potential therapeutic significance of CD47 in esophageal squamous cell carcinoma (ESCC).MethodsImmunohistochemistry was employed to identify the characteristics of CD47 and CD133 in 26 pairs of tumor tissues and adjacent non‐tumor tissues and 136 ESCC tissues. Kaplan‐Meier analysis and Cox proportional hazards models were built for estimating the prognostic values of CD47 and CD133 expression and their combined stemness index. Sphere formation assays were undertaken to explore the effects of CD47 inhibition on primary human ESCC CSCs.ResultsResults conclude that CD47 and CD133 expression is increased in tumor tissues as compared to adjacent non‐tumor tissues. A positive correlation between CD47/CD133 expression and differentiation was found in 136 ESCC patients. Survival analysis indicated that patients with high CD47 or CD133 expression exhibited poor overall survival and progression‐free survival (PFS). The combination of high CD47 and CD133 expression was a reliable independent prognostic factor for both OS (HR = 1.940, 95% CI = 1.399‐2.690, P < 0.0001) and progression‐free survival (HR = 1.883, 95% CI = 1.384‐2.562, P < 0.0001). Notably, CD47+ CD133+ ESCC cells were observed to possess the characteristics of CSCs, and anti‐CD47 treatment veritably eliminated the CSCs pool.ConclusionsThe stemness index determined by the expression of CD47 and CD133 is a promising prognostic predictor, and CD47 is a potential therapeutic target for CSCs in ESCC patients.

Abstract A147: AO-176, a next-generation anti-CD47 antibody, induces immunogenic cell death

Abstract Recent success in cancer immunotherapy has targeted immune checkpoints such as PD-1, PDL-1, and CTLA-4 to enhance the cytotoxic activity of the adaptive T-cell immune response. While the clinical response to these therapies has been dramatic for some, many others have shown partial or even no response highlighting the need for alternative or synergistic approaches that activate innate immunity. Disruption of the interaction between SIRP alpha and CD47, an innate checkpoint inhibitor, using anti-CD47 antibodies, for example, is known to enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages and dendritic cells (DCs) leading to processing and presentation of tumor antigens. Recently, we described AO-176, a next generation anti-CD47 antibody that blocks the CD47/SIRP alpha interaction, induces phagocytosis and causes a direct tumor cell-autonomous death while negligibly binding RBCs. Herein, we characterize the ability of our CD47 antibodies such as AO-176 to induce immunogenic cell death (ICD) and damage-associated molecular patterns (DAMPs) in tumor cells and to potentiate chemotherapy-induced ICD/DAMPs. ICD is a process whereby an agent induces cell surface exposure and release of DAMPs from dying cells which stimulates DCs and adaptive immune responses. Tumor cells were treated in vitro with our CD47 antibodies either alone or in combination with chemotherapeutics followed by assessment of ICD/DAMPs using flow cytometry and biochemical assays. RNAseq was also performed on cells undergoing CD47 antibody mediated ICD/DAMP induction to better understand how CD47 inhibition may regulate ICD. AO-176 and other CD47 antibodies, developed by Arch Oncology, caused mitochondrial stress and loss of outer-membrane integrity, typically observed prior to cells undergoing apoptosis. In addition, CD47 antibody treatment induced a significant ER stress response at the genetic level resulting in the surface exposure of ER chaperone proteins calreticulin, Hsp90, and PDIA3. Concomitantly, our CD47 antibodies increased autophagy and JAK/STAT signaling, which resulted in both ATP and HMGB1 release, respectively. Finally, we demonstrated that in combination, our antibodies potentiated the effects of ICD/DAMP-inducing chemotherapy (e.g., doxorubicin). Here, we describe the unique ability of a specific subset of next generation CD47 antibodies, such as AO-176 to induce ICD/DAMPs. RNAseq analysis of treated cells also revealed alteration of several pathways, including those where DAMPs play a role. In summary, next-generation CD47 antibodies such as AO-176 may provide a novel approach to enhancing the current landscape of checkpoint immunotherapy by enhancing both the innate and adaptive immune responses against tumors. Citation Format: Daniel S. Pereira, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Alun J. Carter, Robyn J. Puro, W. Casey Wilson, Pamela T. Manning, Robert W. Carr. AO-176, a next-generation anti-CD47 antibody, induces immunogenic cell death [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A147.

Inhibited CD47 gene affects the clearance of acute myelogenous leukemia stem cells.

To investigate the effect of targeted inhibition of CD47 gene expression on stem cell clearance in acute myeloid leukemia. After the lentiviral CD47-siRNA was transfected into acute myelogenous leukemia stem cells (LSCs), the proliferative status of acute myelogenous LSCs was detected by cell counting kit-8, and the apoptosis of stem cells of acute myeloid leukemia was detected by annexin/propidium iodide flow assays. The expression of Bcl-2, Bcl-xl, MCL-1, PIK3p110β, and interleukin (IL)-3 in acute myeloid LSCs was detected by Western blot analysis and the activity of protein phosphatase 2A (PP2A) and the protein content of CD96 and CD90 were measured by enzyme-linked immunosorbent assay kits. After transfection of the lentivirus CD47-siRNA into acute myeloid LSCs, compared with the empty vector transfection group (control group), the cell viability of the CD47-siRNA transfection group was decreased, and the apoptosis rate was increased. Furthermore, the antiapoptotic protein Bcl-2, Bcl-xl, and MCL-1 and the content of IL-3 protein, CD96, and CD90 was decreased, whereas the activity of PIK3p110β and PP2A protein was increased. Targeted inhibition of CD47 could inhibit the proliferation of myeloid LSCs, promote apoptosis, mobilize the cells into the cell cycle, and reduce the high expression of immune proteins on the cell surface, therefore providing a theoretical basis for the elimination and eradication of LSCs.

Rrx-001 Is a Phase 3 Small Molecule Dual Inhibitor of CD47 and Sirpα with Activity in Multiple Myeloma

Background:CD47 binds to SIRPα on the surface of macrophages delivering a “do not eat” signal to suppress phagocytosis. To evade macrophage-mediated destruction, CD47 is frequently overexpressed in cancers, including multiple myeloma (MM). CD47 inhibition is an area of intense therapeutic interest with three anti-CD47 monoclonal antibodies (mAbs) currently in clinical trials. However, these mAbs have been associated with severe hematologic toxicities. RRx-001 is a minimally toxic small molecule that dually downregulates CD47 on tumor cells and SIRPα on macrophages and triggers tumor associated macrophage phagocytosis of tumor cells in vitro and in vivo. RRx-001 is entering Phase 3 trials for the treatment of multiple cancer indications.Methods:The effect of RRx-001 on the expression of CD47 and SIRPα on macrophages was evaluated with Western blotting and flow cytometry. An in vitro phagocytotic assay was used to determine whether RRx-001 promoted engulfment of multiple myeloma cells by macrophages. Transcriptional mRNA profiling in murine tumor associated macrophages (TAMs) was performed to analyze the cytokine profile of TAMs in the presence or absence of RRx-001. Finally, myeloma bearing mice were treated with RRx-001 in the presence or absence or absence of clodronate to determine the effect of macrophage depletion on RRx-001 anticancer activity.Results:RRx-001 was shown to downregulate CD47 and SIRPα expression on tumor cells and macrophages, respectively, and to promote the phagocytosis of multiple myeloma tumor cells. RRx-001 also stimulated the production of pro-inflammatory cytokines in TAMs. In tumor bearing mice, depletion of macrophages by clodronate reduced the antitumor effects of RRx-001.Conclusions:RRx-001 is a Phase 3-ready small molecule innate immune checkpoint inhibitor, which triggers tumor associated macrophage phagocytosis of high-expressing CD47 tumor cells. Dual downregulation of CD47 and SIRPα by RRx-001 results in TAM repolarization and phagocytosis of tumor cells. Depletion of macrophages by clodronate in tumor-bearing mice reduced the antitumor effects of RRx-001 and further suggests that the target of RRx-001 is the macrophage. DisclosuresCabrales:EpicentRx Inc: Research Funding. Carter:EpicentRx Inc: Employment. Oronsky:EpicentRx Inc: Employment. Reid:EpicentRx Inc: Employment.

A novel fully human anti-CD47 antibody as a potential therapy for human neoplasms with good safety

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy. However the ubiquitous expression of CD47, especially on the RBC, makes the targeted therapy facing safety risk issues. So, how to balance the safety and efficacy during CD47 inhibition is currently a major question. We had reported an anti-CD47 antibody ZF1 with potent anti-tumor effect. In this study, we further developed and assessed a novel fully human anti-CD47 antibody, AMMS4-G4, derived from ZF1 using affinity maturation. AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4, a humanized anti-CD47 antibody in clinical trial, on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice. Additionally, AMMS4-G4 significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy. In cynomolgus monkeys, AMMS4-G4 was safely administered, was well tolerated at doses of 30 and 60 mg/kg, and did not produce serious adverse events, except for the reversible anemia, which was observed after 3 days and started to recover from 9 days later. Remarkably, it was proved by in vitro assay that Hu5F9-G4 induced RBC hemagglutination which wasn't observed in AMMS4-G4. On the whole, AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.

Anti-CD47 treatment enhances anti-tumor T-cell immunity and improves immunosuppressive environment in head and neck squamous cell carcinoma

ABSTRACTHead and neck squamous cell carcinoma (HNSCC) is considered as an immunosuppressive disease, with impaired tumor-infiltrating T lymphocytes and increased suppressive immune cells. The efficacy of CD47 antibodies in immune checkpoint therapy is not clearly understood in HNSCC. In this study, human tissue microarrays and immunocompetent transgenic mouse models were used to explore the expression of CD47 and the use of CD47 antibodies in HNSCC. We identified overexpression of CD47 in HNSCC as compared with the control normal human tissue and also in HNSCC mouse models. The expression of CD47 also correlated with clinicopathological parameters as well as outcome. Furthermore, inhibition of CD47 delayed tumor growth and improved tumor microenvironment by stimulating effector T cells and decreasing suppressive immune cells and regulating the function of CD11b+ Ly6G+ MDSC. Our data suggest that CD47 blockade may be a potential immunotherapeutic target in human HNSCC.

Acute CD47 Blockade During Ischemic Myocardial Reperfusion Enhances Phagocytosis-Associated Cardiac Repair

Our data suggest that, after a myocardial infarction, integrin-associated protein CD47 on cardiac myocytes is elevated. In culture, increased CD47 on the surface of dying cardiomyocytes impairs phagocytic removal by immune cell macrophages. After myocardial ischemia and reperfusion, acute CD47 inhibition with blocking antibodies enhanced dead myocyte clearance by cardiac phagocytes and also improved the resolution of cardiac inflammation, reduced infarct size, and preserved cardiac contractile function. Early targeting of CD47 in the myocardium after reperfusion may be a new strategy to enhance wound repair in the ischemic heart.

Abstract LB-226: Dual inhibition of CD47 and HER2 to radiosensitize breast cancer cells

Abstract Accumulating studies are aimed to combine immunoregulation with the standard of treatment such as radiotherapy in anti-cancer therapy. A potential cross resistance of tumor cells will be a barrier in furthering the long-term control by such combined treatment. CD47, an immunoglobulin-like transmembrane protein, enables cancer cells to escape the immune surveillance by flagging them with a ‘don’t eat me’ signal, which is recognized by the signal regulatory protein α (SIRPα) on macrophages. Here, we report that CD47 expression is enhanced by radiation in breast cancer cells and in vivo irradiated xenograft tumors via NF-κB regulation. In addition, CD47 and HER2 are co-induced by radiation and CD47 expression is specially enhanced by radiation in HER2-expressing cells. Contrasted with primary tumors, co-expression of CD47 and HER2 is elevated in the recurrent breast tumors. Anti-HER2 treatment inhibits CD47 transcription and protein expression with increased macrophage phagocytosis. Likewise, anti-CD47 not only augments macrophage phagocytosis but also reduces the capacity of tumorsphere formation. Radiation with anti-CD47 synergistically suppresses clonogenic survival and the maximal inhibition was achieved by dual inhibition of CD47 and HER2 with radiation. The synergy of in vivo tumor inhibition is established in mouse breast tumor model via local tumor radiotherapy combined with anti-CD47 treatment. These results demonstrate that the HER2-NF-κB-CD47 loop is responsible the cross-resistance of breast tumor against immune surveillance and radiation. Thus dual inhibition of CD47 and HER2 is a potential target to enhance tumor response to immune-radiotherapy. Funding: This study was partially supported by National Cancer Institute RO1 grant R01CA213830-01 (JJL). Note: This abstract was not presented at the meeting. Citation Format: Demet Candas, Lu Zhang, Cheikh Menaa, Ming Fan, Yanhong Zhang, Lin Liu, Weibing Zhou, Soheila Azghadi, Colleen Sweeney, Rulong Shen, Tzu-yin Lin, Chong-xian Pan, Andrew T. Vaughan, Ji Ming Wang, Hong-Wu Chen, Kit Lam, Lun-Quan Sun, Arta M. Monjazeb, William J. Murphy, Jian Jian Li. Dual inhibition of CD47 and HER2 to radiosensitize breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2017-LB-226

CD47 promotes ovarian cancer progression by inhibiting macrophage phagocytosis

Targeting CD47 efficiently enhances macrophage phagocytosis in both physiological and pathological conditions. Anti-CD47 antibodies have been shown to inhibit the progression of several types of cancer. However, the mechanism of anti-CD47 monoclonal antibody (mAb) treatment remains controversial. In this study, we confirmed that CD47 protein is highly expressed in ovarian cancer, and is correlated with poor clinical characteristics and prognosis. CD47 knockdown in the ovarian cancer cell line, SK-OV-3, promoted phagocytosis by macrophages in vitro and inhibited tumor growth in vivo. These data combined suggest that CD47 inhibition is a potential strategy for cancer treatment. Using an anti-CD47 mAb, we found that CD47 inhibition in both SK-OV-3 cells and primary cancer cells was able to recapitulate our knockdown results and led to an increase in the number of infiltrating macrophages. In addition, the CD133+ tumor initiating cells expressed a high level of CD47, and anti-CD47 mAb treatment was able to trigger the phagocytosis of this cell population. In conclusion, our results indicate that CD47 inhibits macrophage phagocytosis of ovarian cancer cells, and down-regulation of CD47 or inhibiting CD47 by mAb was able to reverse the negative effect. Thus, CD47 antibody therapy may be a promising strategy to treat ovarian cancer.

Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells.

Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Inhibition of CD47 Effectively Targets Pancreatic Cancer Stem Cells via Dual Mechanisms.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with unmet medical need and is strongly promoted by tumor-associated macrophages (TAM). The presence of TAMs is associated with poor clinical outcome, and their overall role, therefore, appears to be protumorigenic. The "don't eat me" signal CD47 on cancer cells communicates to the signal regulatory protein-α on macrophages and prevents their phagocytosis. Thus, inhibition of CD47 may offer a new opportunity to turn TAMs against PDAC cells, including cancer stem cells (CSC), as the exclusively tumorigenic population. We studied in vitro and in vivo the effects of CD47 inhibition on CSCs using a large set of primary pancreatic cancer (stem) cells as well as xenografts of primary human PDAC tissue. CD47 was highly expressed on CSCs, but not on other nonmalignant cells in the pancreas. Targeting CD47 efficiently enhanced phagocytosis of a representative set of primary human pancreatic cancer (stem) cells and, even more intriguingly, also directly induced their apoptosis in the absence of macrophages during long-term inhibition of CD47. In patient-derived xenograft models, CD47 targeting alone did not result in relevant slowing of tumor growth, but the addition of gemcitabine or Abraxane resulted in sustained tumor regression and prevention of disease relapse long after discontinuation of treatment. These data are consistent with efficient in vivo targeting of CSCs, and strongly suggest that CD47 inhibition could be a novel adjuvant treatment strategy for PDAC independent of underlying and highly variable driver mutations.

Cell rigidity and shape override CD47's “self”-signaling in phagocytosis by hyperactivating myosin-II

AbstractA macrophage engulfs another cell or foreign particle in an adhesive process that often activates myosin-II, unless the macrophage also engages “marker of self” CD47 that inhibits myosin. For many cell types, adhesion-induced activation of myosin-II is maximized by adhesion to a rigid rather than a flexible substrate. Here we demonstrate that rigidity of a phagocytosed cell also hyperactivates myosin-II, which locally overwhelms self-signaling at a phagocytic synapse. Cell stiffness is one among many factors including shape that changes in erythropoiesis, in senescence and in diseases ranging from inherited anemias and malaria to cancer. Controlled stiffening of normal human red blood cells (RBCs) in different shapes does not compromise CD47's interaction with the macrophage self-recognition receptor signal regulatory protein alpha (SIRPA). Uptake of antibody-opsonized RBCs is always fastest with rigid RBC discocytes, which also show that maximal active myosin-II at the synapse can dominate self-signaling by CD47. Rigid but rounded RBC stomatocytes signal self better than rigid RBC discocytes, highlighting the effects of shape on CD47 inhibition. Physical properties of phagocytic targets thus regulate self signaling, as is relevant to erythropoiesis, to clearance of rigid RBCs after blood storage, clearance of rigid pathological cells such as thalassemic or sickle cells, and even to interactions of soft/stiff cancer cells with macrophages.

Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration

Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the stroma of many tumours and are frequently associated with the progression of several types of cancer. We investigated the role of M2 macrophages in colon cancer progression and found that human colon cancer tissue had elevated numbers of CD68+ (macrophage marker) cells and CD206+ (M2 macrophage marker) cells and increased CD47 expression. To explore potential interplay between colon cancer cells and M2 macrophages, we differentiated the monocyte cell line THP-1 into M1 and M2 macrophages (CD206high and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced SW480 cell migration and CD47 expression. Factors released by macrophages were involved in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA significantly reduced the migration of SW480 cells in the presence of M2 macrophages. This effect was further decreased via blocking antibodies against the CD47 ligand signal-regulatory protein α (SIRPα). Additionally, cancer cells also secreted significant levels of IL-10, thereby promoting M2 macrophage differentiation. These findings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis.

Abstract 2453: CD47 is a novel therapeutic target for hepatocellular carcinoma

Abstract Hepatocellular Carcinoma (HCC) is a deadly cancer which is chemoresistant and highly recurrent. Tumor-Initiating Cells (T-ICs), which is capable of tumor initiation, self-renewal, differentiation and chemoresistance, may account for the poor prognosis of HCC. Targeting liver T-ICs is hence critical for therapeutic intervention. Using gene expression profiling by cDNA microarray, we found significantly upregulation of CD47 in enriched liver T-ICs compared with their differentiated progenies. CD47 is a membrane protein involved in self-nonself discrimination in immune system. It is also reported to be T-IC markers in Acute Myeloid Leukemia and bladder cancer. The roles of CD47 in immune evasion and T-ICs identification imply CD47 as a potential therapeutic target in HCC. To evaluate if CD47 is a possible liver T-IC marker, we compared CD47 expression in a panel of HCC cell line with non-tumorigenic normal liver cell line (MIHA) by flow cytometry. CD47 expression across HCC cell line panel ranged from 40.6% to 80.1%, with the highest expression in 97L cells. Notably, scanty expression was detected in MIHA. Oncogenomic analysis also documented over-expression of CD47 in HCC clinical samples, revealing the role of CD47 in hepatocarcinogensis. Further investigation of CD47 as T-IC marker was performed by FACS-sorting analysis. CD47+ HCC cells displayed higher tumorigenic ability and exhibited self-renewing phenotypes, including enhanced sphere-forming ability and preferential expression of “stemness’ genes. As CD47+ cells possess T-IC properties, targeting CD47 may be a new therapeutic strategy against HCC. Lentiviral based knockdown was employed to examine effects of CD47 suppression. CD47 knockdown resulted in marked decrease in tumor forming, self-renewal and invasive abilities. To further investigate underlying mechanisms of CD47, cDNA microarray was performed by profiling between CD47 knockdown cells and control cells. Cathepsin S (CTSS), a cysteine protease which is crucial in chemoresistance, was identified as a downstream effecter in CD47 signalling. Interestingly, knockdown of CD47 increased chemo-sensitivity of HCC cells towards chemotherapeutic drugs. These, together with observation that chemoresistant HCC cells exhibited elevated levels of both CD47 and CTSS, suggests CD47 targeting combined with chemotherapy may be an effective treatment option for HCC. Using HCC xenograft tumor model, therapeutic role of CD47 and its combined effect with chemotherapy were assessed in vivo. Suppression of CD47 expression by vivo-morpholino alone caused tumor shrinkage while combination of CD47 targeting with doxorubicin exhibited a synergistic effect in tumor suppression. In summary, our findings supported that CD47 inhibition suppressed self-renewal, tumor initiating ability and chemoresistance of dormant liver T-ICs. Our findings may have important ramifications in future therapeutics against this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2011-2453