Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients.

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.

Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent.

Programmed cell death-1 (PD-1)-targeted therapies enhance T cell responses and show efficacy in multiple cancers, but the role of costimulatory molecules in this T cell rescue remains elusive. Here, we demonstrate that the CD28/B7 costimulatory pathway is essential for effective PD-1 therapy during chronic viral infection. Conditional gene deletion showed a cell-intrinsic requirement of CD28 for CD8 T cell proliferation after PD-1 blockade. B7-costimulation was also necessary for effective PD-1 therapy in tumor-bearing mice. In addition, we found that CD8 T cells proliferating in blood after PD-1 therapy of lung cancer patients were predominantly CD28-positive. Taken together, these data demonstrate CD28-costimulation requirement for CD8 T cell rescue and suggest an important role for the CD28/B7 pathway in PD-1 therapy of cancer patients.

Association of Cytoplasmic CXCR4 With Loss of Epithelial Marker and Activation of ERK1/2 and AKT Signaling Pathways in Non-Small-Cell Lung Cancer.

OBJECTIVES: Compelling evidence demonstrates that CXC-chemokine receptor 4 (CXCR4) is involved in tumor invasion, angiogenesis, metastasis, and resistance to chemotherapy in addition to being one of the coreceptors for T-tropic human immunodeficiency virus entry into T cells. However, it remains controversial as to how to identify functionally activated CXCR4 in tumor biopsies, which would assist in determining which patients may benefit from potential CXCR4-targeted therapy. MATERIALS AND METHODS: Immunohistochemistry (IHC) staining on archival tissues of patients with non-small-cell lung cancer (NSCLC) was used to detect a panel of biomarkers, including phospho-ERK1/2, phospho-AKT, and E-cadherin, which are relevant to downstream signaling of CXCR4 and epithelial to mesenchymal transition (EMT). We also examined whether subcellular localization of CXCR4 could help define possible activation of CXCR4. RESULTS: A total of 94 primary tumor tissue samples from patients with NSCLC were included. Sixty-six patients had both cytomembranous and nuclear staining of CXCR4, 22 had solely nuclear staining, 5 had solely cytomembranous staining, and 1 had negative staining. Cytoplasmic location of CXCR4 with or without nuclear location was associated with loss of the epithelial marker E-cadherin (P = .0015) and activation of ERK1/2 (P = .0121) and AKT (P = .0024), suggesting EMT in these tumors; whereas tumors with only nuclear location of CXCR4 were more indolent and preserved an epithelial phenotype. CONCLUSIONS: Our study suggests that different subcellular localization of CXCR4 may be associated with different activation states; cytoplasmic CXCR4 seems to correlate with biomarker changes associated with EMT in NSCLC.

Honokiol enhances paclitaxel efficacy in multi-drug resistant human cancer model through the induction of apoptosis.

Resistance to chemotherapy remains a major obstacle in cancer therapy. This study aimed to evaluate the molecular mechanism and efficacy of honokiol in inducing apoptosis and enhancing paclitaxel chemotherapy in pre-clinical multi-drug resistant (MDR) cancer models, including lineage-derived human MDR (KB-8-5, KB-C1, KB-V1) and their parental drug sensitive KB-3-1 cancer cell lines. In vitro analyses demonstrated that honokiol effectively inhibited proliferation in KB-3-1 cells and the MDR derivatives (IC50 ranging 3.35 ± 0.13 µg/ml to 2.77 ± 0.22 µg/ml), despite their significant differences in response to paclitaxel (IC50 ranging 1.66 ± 0.09 ng/ml to 6560.9 ± 439.52 ng/ml). Honokiol induced mitochondria-dependent and death receptor-mediated apoptosis in MDR KB cells, which was associated with inhibition of EGFR-STAT3 signaling and downregulation of STAT3 target genes. Combined treatment with honokiol and paclitaxel synergistically augmented cytotoxicity in MDR KB cells, compared with treatment with either agent alone in vitro. Importantly, the combined treatment significantly inhibited in vivo growth of KB-8-5 tumors in a subcutaneous model. Tumor tissues from the combination group displayed a significant inhibition of Ki-67 expression and an increase in TUNEL-positive cells compared with the control group. These results suggest that targeting multidrug resistance using honokiol in combination with chemotherapy drugs may provide novel therapeutic opportunities.

RRM2 regulates Bcl-2 in head and neck and lung cancers: a potential target for cancer therapy.

PURPOSE: Ribonucleotide reductase subunit M2 (RRM2) plays an active role in tumor progression. Recently, we reported that depletion of RRM2 by systemic delivery of a nanoparticle carrying RRM2-specific siRNA suppresses head and neck tumor growth. The aim of this study is to clarify the underlying mechanism by which RRM2 depletion inhibits tumor growth. EXPERIMENTAL DESIGN: siRNA-mediated gene silencing was carried out to downregulate RRM2. Immunoblotting, reverse-transcriptase PCR, confocal microscopy, tissue fractionation, gene overexpression and knockdown were employed to analyze critical apoptosis signaling. Conventional immunohistochemistry and quantum dot-based immunofluorescence were applied to detect RRM2 and Bcl2 expression and localization in tissue samples from patients and mice. RESULTS: Knockdown of RRM2 led to apoptosis through the intrinsic pathway in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines. We showed that Bcl-2 is a key determinant controlling apoptosis, both in vitro and in vivo, and that RRM2 depletion significantly reduces Bcl-2 protein expression. We observed that RRM2 regulates Bcl-2 protein stability, with RRM2 suppression leading to increased Bcl-2 degradation, and identified their colocalization in HNSCC and NSCLC cells. In a total of 50 specimens each from patients with HNSCC and NSCLC, we identified the colocalization of Bcl-2 and RRM2 and found a significant positive correlation between their expression in HNSCC (R = 0.98; P < 0.0001) and NSCLC (R = 0.92; P < 0.0001) tumor tissues. CONCLUSIONS: Our novel findings add to the knowledge of RRM2 in regulating expression of the antiapoptotic protein Bcl-2 and reveal a critical link between RRM2 and Bcl-2 in apoptosis signaling.

Disruption of CHTF18 causes defective meiotic recombination in male mice.

CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.

The pivotal role of integrin ß1 in metastasis of head and neck squamous cell carcinoma.

PURPOSE: This study aimed to understand the prognostic value of integrin ß1 expression in head and neck squamous cell carcinoma (HNSCC) and the mechanism underlying its association with metastatic HNSCC. EXPERIMENTAL DESIGN: Archival HNSCC tissues including 99 nonmetastatic primary tumors and 101 metastatic primary tumors were examined for the association of integrin ß1 expression with metastasis and disease prognosis by appropriate statistical methods. Fluorescence-activated cell sorting was used to separate the integrin ß1(high/+) cell population from the integrin ß1(low/-) population in HNSCC cell lines. These two populations and integrin ß1 shRNA knockdown HNSCC cells were examined for the effect of integrin ß1 on invasion in vitro and on lymph node and lung metastases in a xenograft mouse model. Expression and activation of matrix metalloproteinases (MMP) were examined by zymography. RESULTS: Statistical analysis showed that integrin ß1 expression was significantly higher in the metastatic primary tumors than in the nonmetastatic tumors (42.6% vs. 24.8%, P < 0.0001 and P < 0.0001 by univariate and multivariate analyses, respectively). In patients with lymph node metastasis, integrin ß1 expression was inversely correlated with overall survival (P = 0.035). The integrin ß1 knockdown or integrin ß1(low/-) HNSCC cells showed a significant reduction in lymph node and lung metastases in vivo (P < 0.001 and P < 0.05, respectively). Significantly reduced Matrigel invasion capability was also found in integrin ß1 knockdown or integrin ß1(low/-) HNSCC cells (P < 0.01). Finally, zymography results showed integrin ß1-affected HNSCC invasion by regulating MMP-2 activation. CONCLUSION: These findings indicate that integrin ß1 has a major impact on HNSCC prognosis through its regulation of metastasis.

Systemic delivery of siRNA nanoparticles targeting RRM2 suppresses head and neck tumor growth.

Systemic delivery of siRNA to solid tumors remains challenging. In this study, we investigated the systemic delivery of a siRNA nanoparticle targeting ribonucleotide reductase subunit M2 (RRM2), and evaluated its intratumoral kinetics, efficacy and mechanism of action. Knockdown of RRM2 by an RNAi mechanism strongly inhibited cell growth in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines. In a mouse xenograft model of HNSCC, a single intravenous injection led to the accumulation of intact nanoparticles in the tumor that disassembled over a period of at least 3days, leading to target gene knockdown lasting at least 10days. A four-dose schedule of siRNA nanoparticle delivering RRM2 siRNA targeted to HNSCC tumors significantly reduced tumor progression by suppressing cell proliferation and inducing apoptosis. These results show promise for the use of RRM2 siRNA-based therapy for HNSCC and possibly NSCLC.

Pharmacodynamics of DT-IgG, a dual-targeting antibody against VEGF-EGFR, in tumor xenografted mice.

PURPOSE: DT-IgG is a fully humanized dual-target therapeutic antibody being developed to simultaneously target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), important signaling molecules for tumor growth. The antitumor pharmacodynamics (PD) of DT-IgG was studied in nude mice bearing human tumor xenografts with different EGFR and VEGF expressions and K-ras oncogene status and compared with bevacizumab, cetuximab and bevacizumab + cetuximab. METHODS: Mice bearing human oral squamous cell carcinoma (Tu212), lung adenocarcinoma (A549), or colon cancer (GEO) subcutaneous xenografts were administered with the antibodies intraperitoneally (i.p.), and tumor volumes were measured versus time. Nonlinear mixed effects modeling (NONMEM) was used to study drug potencies (IC(50)) and variations in tumor growth. RESULTS: The PD models adequately described tumor responses for the antibody dose regimens. In vivo IC(50) values varied with EGFR and K-ras status. DT-IgG had a similar serum t (1/2) as cetuximab (~1.7 vs. 1.5 day), was more rapid than bevacizumab (~6 day), and had the largest apparent distribution volume (DT-IgG > cetuximab > bevacizumab). The efficacy of DT-IgG was comparable to bevacizumab despite lower serum concentrations, but was less than bevacizumab + cetuximab. CONCLUSIONS: A lower IC(50) of DT-IgG partially compensated for lower serum concentrations than bevacizumab and cetuximab, but may require higher doses for comparable efficacy as the combination. The model adequately predicted variations of tumor response at the DT-IgG doses tested and could be used for targeting specific tumor efficacies for future testing.

A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment.

An antibody simultaneously targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), two major tumor growth-driving machineries, may provide a novel effective strategy for optimizing tumor targeting and maximizing potential clinical benefits. Human domain antibodies selected against VEGF and EGFR were formatted into a fully human dual-targeting IgG (DT-IgG) to directly target both antigens in a single molecule. We evaluated the efficacy of DT-IgG in comparison with bevacizumab and cetuximab alone and in combination in the lung cancer cell line A549 (low EGFR expression and KRAS mutant) and the head and neck squamous cell carcinoma (HNSCC) cell line Tu212 (high EGFR expression and KRAS wild type) in vitro and in vivo. DT-IgG suppressed Tu212 and A549 cell growth, inhibited EGFR activation and induced apoptosis as effectively as cetuximab, and neutralized VEGF as effectively as bevacizumab. DT-IgG induced EGFR-dependent VEGF internalization, constituting a novel antiangiogenesis mechanism. In xenograft models with lung and head and neck cancer cell lines, DT-IgG displayed efficacy equivalent to bevacizumab in diminishing tumor growth despite its short serum half-life (36 hr in rats) and both agents may constitute preferable alternatives to cetuximab in KRAS-mutant tumors. Immunofluorescence staining revealed that localization of DT-IgG was similar to that of cetuximab, largely associated with EGFR+tumor cells. Our proof of principle study suggests a DT-IgG against EGFR and VEGF as an alternative therapeutic strategy with potentially enhanced clinical benefit.

A folate receptor-targeting nanoparticle minimizes drug resistance in a human cancer model.

Resistance to chemotherapy is a major obstacle in cancer therapy. The main purpose of this study is to evaluate the potential of a folate receptor-targeting nanoparticle to overcome/minimize drug resistance and to explore the underlying mechanisms. This is accomplished with enhanced cellular accumulation and retention of paclitaxel (one of the most effective anticancer drugs in use today and a well-known P-glycoprotein (P-gp) substrate) in a P-gp-overexpressing cancer model. The folate receptor-targeted nanoparticle, HFT-T, consists of a heparin-folate-paclitaxel (HFT) backbone with an additional paclitaxel (T) loaded in its hydrophobic core. In vitro analyses demonstrated that the HFT-T nanoparticle was superior to free paclitaxel or nontargeted nanoparticle (HT-T) in inhibiting proliferation of P-gp-overexpressing cancer cells (KB-8-5), partially due to its enhanced uptake and prolonged intracellular retention. In a subcutaneous KB-8-5 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably prolonged retention within tumor tissues. Importantly, HFT-T treatment markedly retarded tumor growth in a xenograft model of resistant human squamous cancer. Immunohistochemical analysis further indicated that increased in vivo efficacy of HFT-T nanoparticles was associated with a higher degree of microtubule stabilization, mitotic arrest, antiangiogenic activity, and inhibition of cell proliferation. These findings suggest that when the paclitaxel was delivered as an HFT-T nanoparticle, the drug is better retained within the P-gp-overexpressing cells than the free form of paclitaxel. These results indicated that the targeted HFT-T nanoparticle may be promising in minimizing P-gp related drug resistance and enhancing therapeutic efficacy compared with the free form of paclitaxel.

HFT-T, a targeting nanoparticle, enhances specific delivery of paclitaxel to folate receptor-positive tumors.

Nonspecific distribution of chemotherapeutic drugs (such as paclitaxel) is a major factor contributing to side effects and poor clinical outcomes in the treatment of human head and neck cancer. To develop novel drug delivery systems with enhanced efficacy and minimized adverse effects, we synthesized a ternary conjugate heparin-folic acid-paclitaxel (HFT), loaded with additional paclitaxel (T). The resulting nanoparticle, HFT-T, is expected to retain the antitumor activity of paclitaxel and specifically target folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of paclitaxel. In vitro experiments found that HFT-T selectively recognizes FR-positive human head and neck cancer cell line KB-3-1, displaying higher cytotoxicity compared to the free form of paclitaxel. In a subcutaneous KB-3-1 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably improved antitumor efficacy of paclitaxel. The average tumor volume in the HFT-T treatment group was 92.9 +/- 78.2 mm(3) vs 1670.3 +/- 286.1 mm(3) in the mice treated with free paclitaxel. Furthermore, paclitaxel tumors showed a resurgence of growth after several weeks of treatment, but this was not observed with HFT-T. This indicates that HFT-T could be more effective in preventing tumors from developing drug resistance. No significant acute in vivo toxicity was observed. These results indicate that specific delivery of paclitaxel with a ternary structured nanoparticle (HFT-T) targeting FR-positive tumor is a promising strategy to enhance chemotherapy efficacy and minimize adverse effects.