EGFR Oncogenic Mutations in NSCLC Impair Macrophage Phagocytosis and Mediate Innate Immune Evasion Through Up-Regulation of CD47.

INTRODUCTION: EGFR-mutated NSCLC is characterized by an immunosuppressive microenvironment that confers limited clinical effectiveness to anti-PD-1 or PD-L1 antibodies. Despite the discouraging outcomes of immunotherapy, novel immune checkpoints are constantly emerging, among which the specific vulnerability for therapeutic intervention in the context of EGFR-mutated NSCLC remains unresolved. METHODS: Data sets of patient- and cell line-levels were used for screening and mutual validation of association between EGFR mutation and a panel of immune checkpoint-related genes. Regulatory mechanism was elucidated through in vitro manipulation of EGFR signaling pathway and evaluated by immunoblot analysis, quantitative polymerase chain reaction, flow cytometry, immunofluorescence staining, and chromatin immunoprecipitation. In vivo investigation of different therapeutic strategies were conducted using both immunocompetent and immunodeficient mouse models. RESULTS: Among all screened immune checkpoints, CD47 emerged as the candidate most relevant to EGFR activation. Mechanistically, EGFR mutation constitutively activated downstream ERK and AKT pathways to respectively up-regulate the transcriptional factors c-Myc and NF-κB, both of which structurally bound to the promotor region of CD47 and actively transcribed this "don't eat me" signal. Impaired macrophage phagocytosis was observed on introduction of EGFR-sensitizing mutations in NSCLC cell line models, whereas CD47 blockade restored the phagocytic capacity and augmented tumor cell killing in both in vitro and in vivo models. Remarkably, the combination of anti-CD47 antibody with EGFR tyrosine kinase inhibitor revealed an additive antitumor activity compared with monotherapy of either antitumor agent in both immunocompetent and adaptive immunity-deficient mouse models. CONCLUSIONS: EGFR-sensitizing mutation facilitates NSCLC's escape from innate immune attack through up-regulating CD47. Combination therapy incorporating CD47 blockade holds substantial promise for clinical translation in developing more effective therapeutic approaches against EGFR-mutant NSCLC.

Tumor-driven like macrophages induced by conditioned media from pancreatic ductal adenocarcinoma promote tumor metastasis via secreting IL-8.

Tumor-associated macrophages (TAMs) are abundant population of inflammatory cells which play an essential role in remodeling tumor microenvironment and tumor progression. Previously, we found the high density of TAMs was correlated with lymph node metastasis and poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Therefore, this study was designed to investigate the mechanisms of interaction between TAMs and PDAC. THP-1 monocytes were the exposure to conditioned media (CM) produced by PDAC cells; then, monocyte recruitment and macrophage differentiation were assessed. CM from PDAC attracted and polarized THP-1 monocytes to tumor-driven like macrophages. mRNA expression cytokine profiling and ELISA identified the IL-8 secretion was increasing in tumor-driven like macrophages, and STAT3 pathway was involved. Addition of exogenous recombinant human IL-8 promoted PDAC cells motility in vitro and metastasis in vivo via upregulating Twist expression, which mediated epithelial-mesenchymal transition in cancer cells. What is more, IL-8 expression level in tumor stroma by immunohistochemical analysis was related to lymph node metastasis, the number of tumor CD68 but not CD163 positive macrophages and patient outcome. Taken together, these findings shed light on the important interplay between cancer cells and TAMs in tumor microenvironment and suggested that IL-8 signaling might be a potential therapeutic target for PDAC.

Bmi1 combines with oncogenic KRAS to induce malignant transformation of human pancreatic duct cells in vitro.

It is critical to understand the pathogenesis of preinvasive stages of pancreatic duct adenocarcinoma (PDAC) for developing novel potential diagnostic and therapeutic targets. The polycomb group family member B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi1) is overexpressed and involved in cancer progression in PDAC; however, its role in the multistep malignant transformation of human pancreatic duct cells has not been directly demonstrated. In this study, we stably expressed Bmi1 in a model of telomerase-immortalized human pancreatic duct-derived cells (HPNE) and showed that Bmi1 promoted HPNE cell proliferation, migration, and invasion but not malignant transformation. We then used mutant KRASG12D as a second oncogene to transform HPNE cells and showed that it further enhanced Bmi1-induced malignant potential. More importantly, coexpression of KRASG12D and Bmi1 caused anchorage-independent growth transformation in vitro but still failed to produce tumors in nude mice. Finally, we found that mutant KRASG12D induced HPNE-Bmi1 cells to undergo partial epithelial-mesenchymal transition (EMT) likely via upregulation of snail. Knockdown of KRASG12D significantly reduced the expression of snail and vimentin at both the messenger RNA (mRNA) and protein level and further impaired the anchorage-independent growth capability of invasive cells. In summary, our findings demonstrate that coexpression of Bmi1 and KRASG12D could lead to transformation of HPNE cells in vitro and suggest potential new targets for diagnosis and treatment of PDAC.

Safety and efficacy of S-1 chemotherapy in recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy: multi-institutional retrospective analysis.

PURPOSE: This retrospective study evaluates the efficacy and safety of S-1 chemotherapy for recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy. PATIENTS AND METHODS: Thirty-nine patients with recurrent and metastatic nasopharyngeal carcinoma who failed previous platinum-based chemotherapy received oral S-1 chemotherapy (twice daily from day 1 to 14) every 3 weeks. The dose of S-1 was determined according to the body surface area (BSA): 40 mg twice a day for BSA <1.25 m(2); 50 mg twice a day for 1.25 m(2) ≤BSA<1.5 m(2); and 60 mg twice a day for BSA ≥1.5 m(2). RESULTS: Treatment was well tolerated. Most adverse events were mild. Grade 3 hematological toxicity occurred in 7.7%. There was one complete response (2.6%) and 12 partial responses (30.7%), giving an overall response rate of 33.3% (95% CI [confidence interval], 21.7-50.8). Median time-to-progression was 5.6 months, and median survival was 13.9 months. One- and 2-year survival rates were 60% and 26%, respectively. CONCLUSION: S-1 monotherapy is considered a safe and effective treatment option for recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy.

Ex vivo induction of anti-leukemia cytotoxic T cell effect by dendritic cells from human umbilical cord blood cell origin.

To explore the possibility of in vitro induction of cord blood cell-derived lymphocytes into cytotoxic T lymphocytes (CTL) with anti-leukemia specificity, umbilical cord blood (UCB)-derived mononuclear cells were cultured with multiple cytokines to generate dendritic cells (DC) in vitro. Leukemia cells were irradiated with (137)Cs and activated by premature cytokines. The characteristics of maturation of DC were evaluated through morphology examination and flow cytometry. DC pulsed with leukemic antigens were co-cultured with lymphocytes. Cytotoxicity of the CTL to corresponding leukemic cells was measured with lactate dehydrogenase-release assay. The results showed that UCB-derived monocytes could be induced into typical DC in all of the 12 samples. Expression of immunological markers such as CD1a(+), HLA-DR(+), CD86(+), CD83(+) on DC were significantly up-regulated (P < 0.05). DC presenting leukemic antigens generated leukemia-specific CTL with a killing rate of (44.76 +/- 17.42)% at the E:T ratio of 50:1 against AML cells and a killing rate of (8.50 +/- 4.25)% at the E:T ratio of 50:1 against ALL cells. Whereas, these CTL present almost no killing effect on the mononuclear cells collected from the same patients in complete remission phase. It is concluded that (1) it is possible to induce UCB-derived monocytes into mature DC with typical morphology. (2) Cord blood derived mature DC presenting leukemia antigen can generate leukemia-specific CTL with vigorous cytotoxic activity against the same leukemia blasts and low killing activity against bone marrow cells of the same patients in complete remission phase.

[In vitro anti-leukemia activity of cord blood original cytotoxic T lymphocytes].

BACKGROUND & OBJECTIVE: Cord blood transplantation (CBT) possesses graft-versus-leukemia (GVL) effect. It is possible to cure leukemia if specific cytotoxic T lymphocytes (CTLs) can be induced from lymphocytes in cord blood (CB), and be used to eradicate minimal residual disease (MRD) in leukemia patients. This study was to induce CTLs from cord blood, and explore its in vitro anti-leukemia activity. METHODS: Dendritic cells (DCs) were generated from 8 samples of cord blood mononuclear cells by culturing with multiple cytokines. Immature DCs were pulsed with apoptotic leukemia cells to present leukemic antigens to cord blood original lymphocytes to obtain CTLs. The characteristics of maturation of DCs were evaluated by morphology and flow cytometry. Anti-leukemia effect of CTLs was measured by lactate dehydrogenase release assay. RESULTS: Typical DCs were induced from all of the 8 samples. Expressions of immunologic markers CD1a(+), HLA-DR(+), CD86(+), CD83(+) on DCs were significantly higher after culturing than before culturing [(29.6+/-13.8)% vs. (1.8+/-1.9)%, (81.1+/-17.8)% vs. (19.4+/-10.6)%, (42.7+/-21.9)% vs. (7.5+/-5.3)%, (8.0+/-6.9)% vs. (1.4+/-1.1)%, respectively, P< 0.05]. When E:T ratio was 50:1, CTLs showed far higher cytotoxicity to uncultured acute myeloid leukemia (AML) cells than to K562 cells, and to mononuclear cells of bone marrow from the corresponding patients in complete remission phase (CR-MNCs) [(52.6+/-21.0)% vs. (18.2+/-20.2)%, and (3.3+/-6.3)%, P < 0.05]. CONCLUSIONS: Mature DCs derived from cord blood, which loaded leukemia antigens, could induce leukemia-specific CTLs. The CTLs have vigorous cytotoxicity to original leukemia cells rather than CR-MNCs.

[The morphological and immunological characteristics of less-differentiated acute myeloid leukemic cells].

The aim was to study the morphological, histochemical and immunological characteristics of less-differentiated acute myeloid leukemic cells, and their diagnostic significance. Wright-Giemsa and histochemical staining were used to stain bone marrow smears from 2 case of AML-Mo. Immunological phenotypes were determined with flow cytometry. The results showed that myeloperoxidase stainings of both cases were negative, PAS was positive with fine particles, CD33/CD13 were positive, CD2/CD3/CD10/CD19/CD22 were negative. It is concluded that morphology, histochemistry and immunological phenotype on bone marrow smears are the main diagnostic basis for AML-Mo. The use of multiple monoclonal antibodies for staining may improve the accuracy.