Immune landscape of oncohistone-mutant gliomas reveals diverse myeloid populations and tumor-promoting function.

Histone H3-mutant gliomas are deadly brain tumors characterized by a dysregulated epigenome and stalled differentiation. In contrast to the extensive datasets available on tumor cells, limited information exists on their tumor microenvironment (TME), particularly the immune infiltrate. Here, we characterize the immune TME of H3.3K27M and G34R/V-mutant gliomas, and multiple H3.3K27M mouse models, using transcriptomic, proteomic and spatial single-cell approaches. Resolution of immune lineages indicates high infiltration of H3-mutant gliomas with diverse myeloid populations, high-level expression of immune checkpoint markers, and scarce lymphoid cells, findings uniformly reproduced in all H3.3K27M mouse models tested. We show these myeloid populations communicate with H3-mutant cells, mediating immunosuppression and sustaining tumor formation and maintenance. Dual inhibition of myeloid cells and immune checkpoint pathways show significant therapeutic benefits in pre-clinical syngeneic mouse models. Our findings provide a valuable characterization of the TME of oncohistone-mutant gliomas, and insight into the means for modulating the myeloid infiltrate for the benefit of patients.

Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis.

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

Single-cell spatial landscape of immunotherapy response reveals mechanisms of CXCL13 enhanced antitumor immunity.

BACKGROUND: Immunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous heterogeneity and mediates both disease progression and response to immune checkpoint inhibitors (ICI). As such, there is an unmet need to elucidate the spatially defined single-cell landscape of the lung cancer microenvironment to understand the mechanisms of disease progression and identify biomarkers of response to ICI. METHODS: Here, in this study, we applied imaging mass cytometry to characterize the tumor and immunological landscape of immunotherapy response in non-small cell lung cancer by describing activated cell states, cellular interactions and neighborhoods associated with improved efficacy. We functionally validated our findings using preclinical mouse models of cancer treated with anti-programmed cell death protein-1 (PD-1) immune checkpoint blockade. RESULTS: We resolved 114,524 single cells in 27 patients treated with ICI, enabling spatial resolution of immune lineages and activation states with distinct clinical outcomes. We demonstrated that CXCL13 expression is associated with ICI efficacy in patients, and that recombinant CXCL13 potentiates anti-PD-1 response in vivo in association with increased antigen experienced T cell subsets and reduced CCR2+ monocytes. DISCUSSION: Our results provide a high-resolution molecular resource and illustrate the importance of major immune lineages as well as their functional substates in understanding the role of the tumor immune microenvironment in response to ICIs.

Single-cell spatial landscapes of the lung tumour immune microenvironment.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1-9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.

Single-cell spatial immune landscapes of primary and metastatic brain tumours.

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell-cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

LncRNA RMST Regulates Neuronal Apoptosis and Inflammatory Response via Sponging miR-150-5p in Parkinson's Disease.

INTRODUCTION: LncRNA rhabdomyosarcoma 2-associated transcript (RMST) serves as a key regulator in neural stem cell fate and is involved in the progression of different neurological diseases. In this research, the serum level and clinical value of RMST in Parkinson's disease (PD) patients were detected, and the underlying mechanism was explored. METHODS: Ninety-nine PD patients and 93 healthy individuals were collected for clinical experiments. SH-SY5Y cells were treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) to establish PD cell models. qRT-PCR was used for the detection of mRNA levels. CCK-8 and flow cytometry were used to detect neuronal viability and apoptosis. The target relationship of RMST with miR-15a-5p was confirmed applying luciferase reporter assay. RESULTS: RMST was present at high levels in both serum of PD patients and PD cell models. Serum RMST had a certain clinical value for the diagnosis of PD with the AUC of 0.892 at a cutoff value of 1.225. Serum RMST was positively associated with the levels of TNF-α (r = 0.421, p < 0.001) and IL-1ß (r = 0.567, p < 0.001) in PD patients. Knockdown of RMST alleviated the apoptosis and inflammatory response of SH-SY5Y cells induced by MPP+. miR-150-5p was the target gene of RMST and less expressed in the clinical serum samples and PD cell models. CONCLUSION: Serum RMST serves as a promising biomarker for the diagnosis of PD. RMST downregulation may regulate the occurrence and development of PD through inhibiting neuron cell apoptosis and the release of inflammatory cytokines via targeting miR-150-5p.

The MNK1/2-eIF4E Axis Supports Immune Suppression and Metastasis in Postpartum Breast Cancer.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.

Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration.

Metastasis is the leading cause of cancer-related deaths, and obesity is associated with increased breast cancer (BC) metastasis. Preclinical studies have shown that obese adipose tissue induces lung neutrophilia associated with enhanced BC metastasis to this site. Here we show that obesity leads to neutrophil-dependent impairment of vascular integrity through loss of endothelial adhesions, enabling cancer cell extravasation into the lung. Mechanistically, neutrophil-produced reactive oxygen species in obese mice increase neutrophil extracellular DNA traps (NETs) and weaken endothelial junctions, facilitating the influx of tumor cells from the peripheral circulation. In vivo treatment with catalase, NET inhibitors or genetic deletion of Nos2 reversed this effect in preclinical models of obesity. Imaging mass cytometry of lung metastasis samples from patients with cancer revealed an enrichment in neutrophils with low catalase levels correlating with elevated body mass index. Our data provide insights into potentially targetable mechanisms that underlie the progression of BC in the obese population.

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4wt overexpression as murine myoblasts expressing FGFR4V550E undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.

A Novel Biodegradable and Thermosensitive Poly(Ester-Amide) Hydrogel for Cartilage Tissue Engineering.

Thermosensitive hydrogels are attractive alternative scaffolding materials for minimally invasive surgery through a simple injection and in situ gelling. In this study, a novel poly(ester-amide) polymer, methoxy poly(ethylene glycol)-poly(pyrrolidone-co-lactide) (mPDLA, P3L7) diblock copolymer, was synthesized and characterized for cartilage tissue engineering. A series of amphiphilic diblock copolymers was synthesized by ring-opening polymerization of mPEG 550, D,L-lactide, and 2-pyrrolidone. By dynamic light scattering analysis and tube-flipped-upside-down method, viscoelastic properties of the mPDLA diblock copolymer solution exhibited sol-gel transition behavior as a function of temperature. An in vitro degradation assay showed that degradation acidity was effectively reduced by introducing the 2-pyrrolidone monomer into the polyester hydrogel. Besides, mPDLA exhibited great biocompatibility in vitro for cell encapsulation due to a high swelling ratio. Moreover, cell viability and biochemical analysis proved that the mPDLA hydrogel presented a great chondrogenic response. Taken together, these results demonstrate that mPDLA hydrogels are promising injectable scaffolds potentially applicable to cartilage tissue engineering.

Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors.

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.

CHCHD2 Is Coamplified with EGFR in NSCLC and Regulates Mitochondrial Function and Cell Migration.

UNLABELLED: Coiled-coil-helix-coiled-coil-helix domain-containing 2, a mitochondrial protein, encoded by CHCHD2 is located at chromosome 7p11.2 and proximal to the EGFR gene. Here, bioinformatic analyses revealed that CHCHD2 is consistently coamplified with EGFR in non-small cell lung carcinoma (NSCLC). In addition, CHCHD2 and EGFR protein expression levels were positively correlated and upregulated relative to normal lung in NSCLC tumor-derived xenografts. Knockdown of CHCHD2 expression in NSCLC cells attenuated cell proliferation, migration, and mitochondrial respiration. CHCHD2 protein-protein interactions were assessed by the complementary approaches of affinity purification mass spectrometry and in vivo proximity ligation. The CHCHD2 interactome includes the apparent hub proteins C1QBP (a mitochondrial protein) and YBX1 (an oncogenic transcription factor), and an overlapping set of hub-associated proteins implicated in cell regulation. IMPLICATIONS: CHCHD2 influences mitochondrial and nuclear functions and contributes to the cancer phenotype associated with 7p11.2 amplification in NSCLC.

Integrated omic analysis of lung cancer reveals metabolism proteome signatures with prognostic impact.

Cancer results from processes prone to selective pressure and dysregulation acting along the sequence-to-phenotype continuum DNA → RNA → protein → disease. However, the extent to which cancer is a manifestation of the proteome is unknown. Here we present an integrated omic map representing non-small cell lung carcinoma. Dysregulated proteins not previously implicated as cancer drivers are encoded throughout the genome including, but not limited to, regions of recurrent DNA amplification/deletion. Clustering reveals signatures composed of metabolism proteins particularly highly recapitulated between patient-matched primary and xenograft tumours. Interrogation of The Cancer Genome Atlas reveals cohorts of patients with lung and other cancers that have DNA alterations in genes encoding the signatures, and this was accompanied by differences in survival. The recognition of genome and proteome alterations as related products of selective pressure driving the disease phenotype may be a general approach to uncover and group together cryptic, polygenic disease drivers.

Daptomycin-loaded polymethylmethacrylate bone cement for joint arthroplasty surgery.

Antibiotic-loaded acrylic bone cement has been frequently used as an infection prophylaxis or antibiotic-loaded spacer in infected arthroplasty. In addition, daptomycin has been used recently against broad spectrum Gram-positive organisms. The goal of this in vitro study is to investigate the bacteriacidal and mechanical properties of daptomycin-incorporated polymethylmethacrylate (PMMA) bone cement and evaluate its feasibility for clinical use. Daptomycin (0.5, 1, or 2 g) was premixed with 40 g of PMMA bone cement powder before curing. The mechanical properties of the daptomycin-loaded acrylic bone cement (DLABC) were estimated following standard guidance, and the release profile and kinetics of daptomycin from PMMA were analyzed. The antimicrobial efficacy of DLABC was determined with a zone of inhibition (ZOI) assay against Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, and Enterococcus faecium, respectively. The results showed that the compressive strength, of PMMA bone cement, which was higher than 100 MPa in all groups, was sufficient according to ISO 5833 after incorporation of daptomycin. The encapsulated daptomycin was released for 2 weeks with a 9.59 ± 0.85%, 15.25 ± 0.69%, and 20.64 ± 20.33% released percentage on the first day in the low, mid, and high groups, respectively. According to the calculated release kinetics, incorporated daptomycin should be 3.3 times the original dose to double its release. Although all recipes of DLABC had a microbial inhibitory effect, the effect with a higher encapsulated amount of daptomycin was more significant. Therefore, we believe that daptomycin can be locally delivered from PMMA bone cement at the surgical site as a prophylactic or treatment for osteomyelitis against Gram-positive organisms with intact cement function.

Proteomic profiles of human lung adeno and squamous cell carcinoma using super-SILAC and label-free quantification approaches.

Nonsmall cell lung cancer (NSCLC) accounts for 85% of lung cancers, and is subdivided into two major histological subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC). There is an unmet need to further subdivide NSCLC according to distinctive molecular features that may be associated with responsiveness to therapies. Four primary tumor-derived xenograft proteomes (two-each ADC and SCC) were quantitatively compared by using a super-SILAC labeling approach together with ultrahigh-resolution MS. Proteins highly differentially expressed in the two subtypes were identified, including 30 that were validated in an independent cohort of 12 NSCLC primary tumor-derived xenograft tumors whose proteomes were quantified by an alternative, label-free shotgun MS methodology. The 30-protein signature contains metabolism enzymes including phosphoglycerate dehydrogenase, which is more highly expressed in SCC, as well as a comprehensive set of cytokeratins and other components of the epithelial barrier, which is therefore distinctly different between ADC and SCC. These results demonstrate the utility of the super-SILAC method for the characterization of primary tissues, and compatibility with datasets derived from different MS-based platforms. The validation of proteome signatures of NSCLC subtypes supports the further development and application of MS-based quantitative proteomics as a basis for precision classifications and treatments of tumors. All MS data have been deposited in the ProteomeXchange with identifier PXD000438 (http://proteomecentral.proteomexchange.org/dataset/PXD000438).

Effect of chondroitin sulphate C on the in vitro and in vivo chondrogenesis of mesenchymal stem cells in crosslinked type II collagen scaffolds.

This study evaluates the crosslinkage effect of chondroitin sulphate C (CSC) and type II collagen (COL II) on chondrogenesis of mesenchymal stem cells (MSCs) in vitro and in vivo. In the in vitro studies, our results show that the weight ratio CSC:COL II that reaches 1.2:100 (CSC1.2/100 -COL II scaffold) can provide an optimal microenvironment for MSC chondrogenesis. When MSCs are cultured in this CSC1.2/100 -COL II scaffold, the chondrogenic gene expression of cultured cells is upregulated, while the osteogenic gene expression of these is downregulated. In addition, MSCs cultivated in the CSC1.2/100 -COL II scaffold are found to express the highest glycosaminoglycans:DNA ratio as compared to those in scaffolds of other CSC:COL II ratios. Histological and immunohistological evidence also supports the result. In the in vivo study, our results show that MSCs cultivated in the CSC1.2/100 -COL II scaffold demonstrate a better repair ability on cartilage lesions than does the COL II scaffold. After 1 month in vivo, the injected MSCs in the CSC1.2/100 -COL II scaffold show lacuna structures and stimulate the formation of type II collagen at the defective sites. Six months after transplantation, the generated cells in the CSC1.2/100 -COL II group show higher gene expressions of type II collagen and aggrecan but lower gene expression of type I collagen at the defective sites than those in the COL II group. The results strongly suggest that CSC1.2/100 -COL II scaffold can serve as a potential candidate for cartilage repair and improve the chondrogenesis of MSCs in general.

Prodigiosin-induced cytotoxicity involves RAD51 down-regulation through the JNK and p38 MAPK pathways in human breast carcinoma cell lines.

RAD51 is essential for homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) in mammalian cells. RAD51 is an attractive target for anticancer drugs, given high RAD51 levels are frequently observed in many human tumors and associated with increased resistance to DSBs-inducing chemotherapeutics. Prodigiosin is a bacterial tripyrrole pigment with potent anticancer activity and also provokes DSBs. We hereby aimed to elucidate the role of RAD51 in prodigiosin-induced cytotoxicity. Prodigiosin was found to down-regulate RAD51 in multiple human breast carcinoma cell lines irrespective of p53 status. Mechanistically, prodigiosin lowered RAD51 mRNA expression, whereas blockade of proteasome-mediated degradation failed to restore RAD51 levels following prodigiosin treatment. In addition, prodigiosin triggered phosphorylation of JNK and p38 MAPK, while pharmacological inhibition of JNK or p38 MAPK attenuated prodigiosin-mediated inhibition of RAD51 mRNA expression. Lastly, cells with enforced RAD51 expression showed increased resistance to prodigiosin-induced cytotoxicity as well as inhibition of colony formation. Collectively, we conclude that RAD51 down-regulation represents one of the modes of prodigiosin's cytotoxic action, ostensibly by augmenting the genotoxic effect of prodigiosin through suppression of RAD51-mediated HR repair. Our findings further implicate the use of prodigiosin to potentiate the cytotoxicity of DSB-inducing chemotherapeutics through RAD51 down-regulation.

Development of natural anti-tumor drugs by microorganisms.

Discoveries of tumor-resistant pharmacological drugs have mainly resulted from screening of natural products and their analogs. Some are also discovered incidentally when studying organisms. The great biodiversity of microorganisms raises the possibility of producing secondary metabolites (e.g., mevastatin, lovastatin, epothilone, salinosporamide A) to cope with adverse environments. Recently, natural plant pigments with anti-tumor activities such as ß-carotene, lycopene, curcumin and anthocyanins have been proposed. However, many plants have a long life cycle. Therefore, pigments from microorganisms represent another option for the development of novel anti-tumor drugs. Prodigiosin (PG) is a natural red pigment produced by microorganisms, i.e., Serratia marcescens and other gram-negative bacteria. The anti-tumor potential of PG has been widely demonstrated. The families of PG (PGs), which share a common pyrrolylpyrromethene (PPM) skeleton, are produced by various bacteria. PGs are bioactive pigments and are known to exert immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. Currently the most common strain used for producing PGs is S. marcescens. However, few reports have discussed PGs production. This review therefore describes the development of an anti-tumor drug, PG, that can be naturally produced by microorganisms, and evaluates the microbial production system, fermentation strategies, purification and identification processes. The application potential of PGs is also discussed.

Primary tumor xenografts of human lung adeno and squamous cell carcinoma express distinct proteomic signatures.

Nonsmall cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. The most prevalent subtypes of NSCLC are adenocarcinoma (ADC) and squamous cell carcinoma (SCC), which combined account for approximately 90%. Ten resected NSCLC patient tumors (5 ADC and 5 SCC) were directly introduced into severely immune deficient (NOD-SCID) mice, and the resulting xenograft tumors were analyzed by standard histology and immunohistochemistry (IHC) and by proteomics profiling. Mass spectrometry (MS) methods involving 1- and 2-dimensional LC-MS/MS, and multiplexed selective reaction monitoring (SRM, or MRM), were applied to identify and quantify the xenograft proteomes. Hierarchical clustering of protein profiles distinguished between the ADC and SCC subtypes. The differential expression of 178 proteins, including a comprehensive panel of intermediate filament keratin proteins, was found to constitute a distinctive proteomic signature associated with the NSCLC subtypes. Epidermal growth factor receptor (EGFR) was expressed in ADC and SCC xenografts, and EGFR network activation was assessed by phosphotyrosine profiling by Western blot analysis and SRM measurement of EGFR levels, and mutation analysis. A multiplexed SRM/MRM method provided relative quantification of several keratin proteins, EGFR and plakophilin-1 in single LC-MS/MS runs. The protein quantifications by SRM and MS/MS spectral counting were associated with superior dynamic range and reproducibility but were otherwise consistent with orthogonal methods including IHC and Western immuno blotting. These findings illustrate the potential to develop a comprehensive MS-based platform in oncologic pathology for better classification and potentially treatment of NSCLC patients.