Background: Treatment multiple tumors by immune therapy can be achieved by mobilizing both innate and adaptive immunity. The programmed death ligand 1 (PD-L1; or CD274, B7-H1) is a critical "don't find me" signal to the adaptive immune system. Equally CD47 is a critical "don't eat me" signal to the innate immune system and a regulator of the adaptive immune response. Method: Both of CD47 and PD-L1 are overexpressed on the surface of cancer cells to enable to escape immunesurveillance. We designed EpCAM (epithelial cell adhesion molecule)-targeted cationic liposome (LPP-P4-Ep) containing si-CD47 and si-PD-L1 could target high-EpCAM cancer cells and knockdown both CD47 and PD-L1 proteins. Findings: Efficient silencing of CD47 and PD-L1 versus single gene silencing in vivo by systemic administration of LPP-P4-Ep could significantly inhibited the growth of solid tumors in subcutaneous and reduced lung metastasis in lung metastasis model. Target delivery of the complexes LPP-P4-Ep increased anti-tumor T cell and NK cell response, and release various cytokines including IFN-γ and IL-6 in vivo and in vitro. Interpretation: This multi-nanoparticles showed significantly high-EpCAM tumor targeting and lower toxicity, and enhanced immune therapeutic efficacy. Our data indicated that dual-blockade tumor cell-specific innate and adaptive checkpoints represents an improved strategy for tumor immunotherapy.

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BACKGROUND: This study was aimed at establishing a sensitive and specific isolation, characterization, and enumeration method for living circulating tumor cells (CTCs) in patients with colorectal carcinoma. METHODS: Quantitative isolation and characterization of CTCs were performed through a combination of immunomagnetic negative enrichment and fluorescence-activated cell sorting. Isolated CTCs were identified by immunofluorescence staining. The viability and purity of the sorted cells were determined by flow cytometry. Blood samples spiked with HCT116 cells (range, 3-250 cells) were used to determine specificity, recovery, and sensitivity. The method was used to enumerate, characterize, and isolate living CTCs in 10 mL of blood from patients with colorectal carcinoma. RESULTS: The average recovery of HCT116 cells was 61% or more at each spiking level, and the correlation coefficient was 0.992. An analysis of samples from all 18 patients with colorectal carcinoma revealed that 94.4% were positive for CTCs with an average of 33 6 24 CTCs per 10 mL of blood and with a diameter of 14 to 20 lm (vs 8-12 lm for lymphoma). All patients were CD47 1 , with only 4.3% to 61.2% being CD44 1 . The number of CTCs was well correlated with the patient TNM stage and could be detected in patients at an early cancer stage. The sorted cells could be recultured, and their viability was preserved. CONCLUSIONS: This method provides a novel technique for highly sensitive and specific detection and isolation of CTCs in patients with colorectal carcinoma. This method complements the existing approaches for the de novo functional identification of a wide variety of CTC types. It is likely to help in predicting a patient's disease progression and potentially in selecting the appropriate treatment. Cancer 2015;121:3036-45.

There are disease-causing biohazards in the blood that cannot be treated with modern medicines. Here we show that an intelligently designed safe biomaterial can precisely identify, tow and dump a targeted biohazard from the blood into the small intestine. Positively charged mesoporous silica nanoparticles (MSNs) functionalized with EGFR-targeting aptamers (MSN-AP) specifically recognize and bind blood-borne negatively charged oncogenic exosomes (A-Exo), and tow A-Exo across hepatobiliary layers and Oddi’s sphincter into the small intestine. MSN-AP specifically distinguish and bind A-Exo from interfering exosomes in cell culture and rat and patient blood to form MSN-AP and A-Exo conjugates (MSN-Exo) that transverse hepatocytes, cholangiocytes, and endothelial monolayers via endocytosis and exocytosis mechanisms, although Kupffer cells have been shown to engulf some MSN-Exo. Blood MSN-AP significantly decreased circulating A-Exo levels, sequentially increased intestinal A-Exo and attenuated A-Exo-induced lung metastasis in mice. This study opens an innovative avenue to relocate blood-borne life-threatening biohazards to the intestine.

Carcinoma metastasis is triggered by a subpopulation of circulating tumor cells (CTCs). And single immune checkpoint therapy is not good enough to inhibit CTC-induced metastasis. Here, we demonstrate that simultaneously blocking CD274 (programmed death ligand 1, PD-L1 or B7-H1) and CD47 checkpoints which were respectively signal of “don’t find me” and “don’t eat me” on CTCs by corresponding antibodies could enhance the inhibition tumor growth than single CD274 or CD47 antibody alone. In vitro flow cytometry data proved that CD47 and CD274 were overexpressed on the tested mouse tumor cell lines. The antibodies could effectively block the expressions of CD47 and CD274 on the cell surface and stably attached to tumor cell surface for several hours. The simultaneous blockade on both CD47 and CD274 checkpoints inhibited tumor growth and CTCs metastasis more potently than a single antibody inhibition or blank control on 4T1 tumor mouse model in vivo. Our results demonstrated that simultaneous dual targeting immune checkpoints, i.e., CD47 and CD274, by using specific antibodies may be more effective as an immunotherapeutics on CTCs than a CD47 or CD274 alone.

The success of cancer immunotherapy on recognition checkpoints for killing cancer cells has raised a great interest of scientists in understanding new and old methods of immunotherapeutic. CD47 (cluster of differentiation 47) is a cell surface glycoprotein and widely expressed on cells, which belongs to the immunoglobulin (Ig) superfamily as a cell membrane receptor which serves in immune therapy. CD47 is an inhibitory receptor expressed on tumor cell surface and interacts with signal receptor protein-alpha (SIPR-α, also named CD172a or SHPS-1) which may escape from immune cells such as macrophage and T cells. Meanwhile, tumor cells express high CD47 protein which may secrete exosomes with high CD47 expression. The high CD47 expression-exosomes could serve the tumor metastasis process and provide transfer convenience for tumors on the microenvironment. CD47 on cancer cells can also affect the migration and invasion of cells. The high CD47 expression on tumor or CTC (circulating tumor cell) surface means the stronger migration and invasion and makes them escape from immune cells for phagocytosis such as T cells, NK (natural killer) cells and macrophage, which could be used for diagnosis and prognosis on cancer patients. Meanwhile, targeting CD47 combined with other biomarkers such as EpCAM (epithelial cell adhesion molecule), CD44, etc on cancer surface could be used to isolate CTCs from patients’ blood. In terms of treatment, anti-CD47 antibody combined with another antibody such as anti-PD-L1 (programmed death-ligand 1) antibody or drugs such as rituximab, DOX or oxaliplatin also has better therapeutic effects and antitumor function to tumors. Using nanomaterials as an intermediary for CD47-related immune therapy could greatly increase the therapeutic effect and overcome multiple biological barriers for anti-CD47 antibody in vivo. In this review, we discuss the important role and the function of CD47 in tumor metastasis and also provide a reference for related research.

Rare circulating tumor cells (CTCs) cause >50% of primary colorectal cancer survivors to develop deadly metastasis at 3-5 years after surgery; the current chemotherapies can do nothing about these cells. Herein, we synthesized a novel doxorubicin (DOX)-entrapped mesoporous silica nanoparticle (MSN), covalently-conjugated with two aptamers, for simultaneously targeting EpCAM and CD44, the typical surface biomarkers of colorectal CTCs. The nanomissile can specifically capture the metastasis-prone CTCs spiked in healthy human blood in a competitive-binding manner. The binding not only accurately delivers DOX into the cancer cells via the biomarker-mediated endocytosis to inhibit CTC viability through the DOX-dependent mechanism, but also inhibits the adhesion of cancer cells to the endothelium and the consequent transmembrane migration through the DOX-independent mechanism. The molecular entity of the conjugate and its pharmaceutical DOX encapsulation-releasing capacity are well-demonstrated via various physiochemical characterizations including gel electrophoresis, which proves the >8-hour biostability of the nanomissile in blood, long enough for it to chase CTCs in mice and synergistically inhibit the CTC-induced lung metastasis more potently than its single aptamer-conjugated counterparts and DOX itself. The present strategy may pave a new avenue for safe and effective cancer metastasis chemoprevention.

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