Surgical resection remains the most effective therapy for solid tumors worldwide. The most important prognostic indicator for cure following cancer surgery is a complete resection with no residual disease. However, intraoperative detection of retained cancer cells after surgery is challenging, and residual disease continues to be the most common cause of local failure. We hypothesized visual enhancement of tumors using near-infrared imaging could potentially identify tumor deposits in the wound after resection.
A small animal model of surgery and retained disease was developed. Residual tumor deposits in the wound were targeted using an FDA approved imaging agent, indocyanine green, by the enhanced permeability and retention (EPR) effect. A novel hand-held spectrometer was used to optically visualize retained disease after surgery.
We found residual disease using near-infrared imaging during surgery that was not visible to the naked eye or microCT. Furthermore, examination of tumor nodules was remarkably precise in delineating margins from normal surrounding tissues. This approach was most successful for tumors with increased neovasculature.
The results suggest that near-infrared examination of the surgical wound after curative resection can potentially enable the surgeon to locate residual disease. The data in this study is the basis of an ongoing Phase I/II clinical trial in patients who undergo resection for lung and breast cancer.
intraoperative imaging; surgical oncology; infrared; margins; indocyanine green
Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.
BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.
Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).
These studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.
Malignant mesothelioma; Tumor immunology; Immune suppression; TGF-β; CD8+ Cytotoxic T cell
Up to 30% of cancer patients undergoing curative surgery develop local recurrences due to positive margins. Patients typically receive adjuvant chemotherapy, immunotherapy and/or radiation to prevent such relapses. Interestingly, evidence supporting these therapies is traditionally derived in animal models of primary tumors, thus failing to consider surgically induced tumor microenvironment changes that may influence adjuvant therapy efficacy. To address this consideration, we characterized a murine model of local cancer recurrence. This model was reproducible and generated a postoperative inflammatory tumor microenvironment that resembles those observed following human cancer surgery. To further validate this model, antagonists of two pro-inflammatory mediators, TGFβ and COX-2, were tested and found to be effective in decreasing the growth of recurrent tumors. We appreciated that preoperative TGFβ inhibition led to wound dehiscence, while postoperative initiation of COX-2 inhibition resulted in a loss of efficacy. In summary, although not an exact replica of all human cancer surgeries, our proposed local recurrence approach provides a biologically relevant and reliable model useful for preclinical evaluation of novel adjuvant therapies. The use of this model yields results that may be overlooked using traditional preclinical cancer models that fail to incorporate a surgical component.
Surgical model; adjuvant therapy; cancer recurrence; immunotherapy; oncology; tumor microenvironment
Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.
salinomycin; endoplasmic reticulum stress; MTOR; autophagy; apoptosis
The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy.
Myeloid derived suppressor cells; docetaxol; RNA aptamer; CpG oligodeoxynucleotides (ODN); cyclophosphamide; gemcitabine; curcumin
The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.
Multiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios.
To test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays.
AdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations.
This study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.
Surgical oncology; Immunotherapy; Cancer; Animal model
Nearly 30% of cancer patients undergoing curative surgery succumb to distant recurrent disease. Despite large implications and known differences between primary and recurrent tumors, preclinical adjuvant therapy evaluation frequently occurs only in primary tumors and not recurrent tumors. We hypothesized that well characterized and reproducible models of postoperative systemic recurrences should be used for preclinical evaluation of adjuvant approaches.
We examined traditional animal models of cancer surgery that generate systemic cancer recurrences. We also investigated models of systemic cancer recurrences that incorporate spontaneously metastatic cell lines and surgical resection. For each model, we critiqued feasibility, reproducibility and similarity to human recurrence biology. Using our novel model, we then tested the adjuvant use of a novel systemic inhibitor of TGF-β, 1D11.
Traditional surgical models are confounded by immunologic factors including concomitant immunity and perioperative immunosuppression. A superior preclinical model of postoperative systemic recurrences incorporates spontaneously metastatic cell lines and primary tumor excision. This approach is biologically relevant and readily feasible. Using this model, we discovered that “perioperative” TGF-β blockade has strong anti-tumor effects in the setting of advanced disease that would not be appreciated in primary tumor cell lines or other surgical models.
There are multiple immunologic effects that rendered previous models of postoperative cancer recurrences inadequate. Use of spontaneously metastatic cell lines followed by surgical resection eliminates these confounders, and best resembles the clinical scenario. This preclinical model provides more reliable preclinical information when evaluating new adjuvant therapies.
Surgery; recurrence; models; surgical oncology; concomitant immunity; perioperative immunosuppression; TGF-β
Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a handheld spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence or surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2–5 × 10−11 M for the indocyanine dye and 0.5–1 × 10−13 M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5–10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.
Optical spectroscopy; fluorescence; surface-enhanced Raman scattering (SERS); nanoparticles; image-guided surgery; surgical oncology; indocyanine green (ICG)
Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.
With the advent of targeted therapies directed towards folate receptor alpha, with several such agents in late stage clinical development, the sensitive and robust detection of folate receptor alpha in tissues is of importance relative to patient selection and perhaps prognosis and prediction of response. The goal of the present study was to evaluate the expression of folate receptor alpha in non-small cell lung cancer specimens to determine its frequency of expression and its potential for prognosis. The distribution of folate receptor alpha expression in normal tissues as well as its expression and relationship to non-small cell lung cancer subtypes was assessed by immunohistochemistry using tissue microarrays and fine needle aspirates and an optimized manual staining method using the recently developed monoclonal antibody 26B3. The association between folate receptor alpha expression and clinical outcome was also evaluated on a tissue microarray created from formalin fixed paraffin embedded specimens from patients with surgically resected lung adenocarcinoma. Folate receptor alpha expression was shown to have a high discriminatory capacity for lung adenocarcinomas versus squamous cell carcinomas. While 74% of adenocarcinomas were positive for folate receptor alpha expression, our results found that only 13% of squamous cell carcinomas were FRA positive (p<0.0001). In patients with adenocarcinoma that underwent surgical resection, increased folate receptor alpha expression was associated with improved overall survival (Hazard Ratio 0.39, 95% CI 0.18-0.85). These data demonstrate the diagnostic relevance of folate receptor alpha expression in non-small cell lung cancer as determined by immunohistochemistry and suggest that determination of folate receptor alpha expression provides prognostic information in patients with lung adenocarcinoma.
folate receptor alpha; FRA; non-small cell lung cancer; adenocarcinoma; prognosis; immunohistochemistry
Rationale: Patients on mechanical ventilation who exhibit diaphragm inactivity for a prolonged time (case subjects) develop decreases in diaphragm force-generating capacity accompanied by diaphragm myofiber atrophy.
Objectives: Our objectives were to test the hypotheses that increased proteolysis by the ubiquitin-proteasome pathway, decreases in myosin heavy chain (MyHC) levels, and atrophic AKT-FOXO signaling play major roles in eliciting these pathological changes associated with diaphragm disuse.
Methods: Biopsy specimens were obtained from the costal diaphragms of 18 case subjects before harvest (cases) and compared with intraoperative specimens from the diaphragms of 11 patients undergoing surgery for benign lesions or localized lung cancer (control subjects). Case subjects had diaphragm inactivity and underwent mechanical ventilation for 18 to 72 hours, whereas this state in controls was limited to 2 to 4 hours.
Measurements and Main Results: With respect to proteolysis in cytoplasm fractions, case diaphragms exhibited greater levels of ubiquitinated-protein conjugates, increased activity of the 26S proteasome, and decreased levels of MyHCs and α-actin. With respect to atrophic signaling in nuclear fractions, case diaphragms exhibited decreases in phosphorylated AKT, phosphorylated FOXO1, increased binding to consensus DNA sequence for Atrogin-1 and MuRF-1, and increased supershift of DNA-FOXO1 complexes with specific antibodies against FOXO1, as well as increased Atrogin-1 and MuRF-1 transcripts in whole myofiber lysates.
Conclusions: Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.
AKT; FOXO; ubiquitination; proteasome; myosin heavy chains
The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).
In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.
This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein–1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non–small-cell lung cancer (NSCLC). Anti-murine–CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8+ T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
tumor immunology; CCL2; lung cancer; mesothelioma; tumor-associated macrophages
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
Surgery is currently the most effective and widely used procedure in treating human cancers, and the single most important predictor of patient survival is a complete surgical resection. Major opportunities exist to develop new and innovative technologies that could help the surgeon to delineate tumor margins, to identify residual tumor cells and micrometastases, and to determine if the tumor has been completely removed. Here we discuss recent advances in nanotechnology and optical instrumentation, and how these advances can be integrated for applications in surgical oncology. A fundamental rationale is that nanometer-sized particles such as quantum dots and colloidal gold have functional and structural properties that are not available from either discrete molecules or bulk materials. When conjugated with targeting ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target malignant tumor cells and tumor microenvironments with high specificity and affinity. In the “mesoscopic” size range of 10–100 nm, nanoparticles also have large surface areas for conjugating to multiple diagnostic and therapeutic agents, opening new possibilities in integrated cancer imaging and therapy.
quantum dots; Raman scattering; molecular probes; intraoperative imaging; cancer; instrumentation
Functional genomics has emerged over the past ten years as a novel technology to study genetic alterations. Gene expression arrays are one genomic technique employed to discover changes in the DNA expression that occur in neoplastic transformation. Microarrays have been applied to investigating lung cancer. Specific applications include discovering novel genetic changes that occur in lung tumors. Microarrays can also be applied to improve diagnosis, staging, and discover prognostic markers. The eventual goal of this technology is to discover new markers for therapy and to customize therapy based on an individual tumor genetic composition. In this review, we present the current state of gene expression array technology in its application to lung cancer.
Lung cancer; genomics; gene expression arrays; gene expression profiling; diagnosis; staging; prognosis; treatment; therapy; management
Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life. We profiled gene expression in human fetal lung epithelial cells cultured in serum-free medium containing dexamethasone and cyclic AMP, a treatment that induces differentiation of type II cells. Microarray analysis identified 388 genes that were induced > 1.5-fold by 72 h of hormone treatment. Induced genes represented all categories of molecular function and subcellular location, with increased frequency in the categories of ionic channel, cell adhesion, surface film, lysosome, extracellular matrix, and basement membrane. In time-course experiments, self-organizing map analysis identified a cluster of 17 genes that were slowly but highly induced (5- to ∼ 190-fold) and represented four functional categories: surfactant-related (SFTPC, SFTPA, PGC, SFTPB, LAMP3, LPL), regulatory (WIF2, IGF2, IL1RL1, NR4A2, HIF3A), metabolic (MAOA, ADH1B, SEPP1), and transport (SCNN1A, CLDN18, AQP4). Induction of both mRNA and protein for these genes, which included nine newly identified regulated genes, was confirmed, and cellular localization was determined in both fetal and postnatal tissue. Induction of lysosomal-associated membrane protein 3 required both hormones, and expression was localized to limiting membranes of lamellar bodies. Hormone-induced differentiation of human type II cells is associated with genome-wide increased expression of genes with diverse functions.
cyclic AMP; epithelial differentiation; glucocorticoid; human fetal lung; type II cell