Unlocking the Potential of Therapy-Induced Cytokine Responses: Illuminating New Pathways in Cancer Precision Medicine.

Precision cancer medicine primarily aims to identify individual patient genomic variations and exploit vulnerabilities in cancer cells to select suitable patients for specific drugs. These genomic features are commonly determined by gene sequencing prior to therapy, to identify individuals who would be most responsive. This precision approach in cancer therapeutics remains a powerful tool that benefits a smaller pool of patients, sparing others from unnecessary treatments. A limitation of this approach is that proteins, not genes, are the ultimate effectors of biological functions, and therefore the targets of therapeutics. An additional dimension in precision medicine that considers an individual's cytokine response to cancer therapeutics is proposed. Cytokine responses to therapy are multifactorial and vary among individuals. Thus, precision is dictated by the nature and magnitude of cytokine responses in the tumor microenvironment exposed to therapy. This review highlights cytokine responses as modules for precision medicine in cancer therapy, including potential challenges. For solid tumors, both detectability of cytokines in tissue fluids and their being amenable to routine sensitive analyses could address the difficulty of specimen collection for diagnosis and monitoring. Therefore, in precision cancer medicine, cytokines offer rational targets that can be utilized to enhance the efficacy of cancer therapy.

Immune Profile of Exosomes in African American Breast Cancer Patients Is Mediated by Kaiso/THBS1/CD47 Signaling.

African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.

Interference with pathways activated by topoisomerase inhibition alters the surface expression of PD-L1 and MHC I in colon cancer cells.

Topoisomerase inhibitors are clinically used to treat various cancer types, including colorectal cancer. These drugs also activate signaling pathways that modulate cell survival and immune cell functions. Immunotherapy is promising for certain tumors, including microsatellite instable colorectal cancer, but not for microsatellite stable colorectal cancer. The reasons for this lack of responsiveness are largely unknown. Understanding how colorectal cancer cell-surface proteins interact with tumor-resident immune cells may offer an opportunity to identify molecules that, if targeted, may render tumor cells visible to immune cells. The present study used flow cytometry, fluorescent staining and immunoblotting to examine if inhibition of pathways activated by topoisomerase-targeting drugs may modulate the outcomes of treatment through effects on cell cycle arrest and apoptosis, and by altering surface expression levels of programmed death-ligand 1 (PD-L1) or major histocompatibility complex protein I (MHC I). Inhibition of either NF-κB or DNA-damage response (DDR) potently enhanced cell death in combination with topoisomerase inhibition, while only NF-κB inhibition increased MHC I. PD-L1 upregulation was moderately affected by NF-κB or DDR inhibitors, while both topoisomerase inhibitors and DNA damaging agents may enhance the surface expression of MHC I molecules on colon cancer cells. Such enhanced expression of MHC I may be suppressed by inhibitors of ataxia-telangiectasia mutated or checkpoint kinase kinases. Additionally, adaptive tolerance to topoisomerase inhibition caused altered cell cycle response, and reduced the expression levels of both PD-L1 and MHC I on both microsatellite instable and stable colon cancer cell lines. Therefore, targeted modulation of DDR pathways, PD-L1, MHC I or other immune regulators in colon cancer cells may make them more visible to immune cells and enable rational combination of conventional therapy with immunotherapy.

MicroRNAs within the Basal-like signature of Quadruple Negative Breast Cancer impact overall survival in African Americans.

We previously found that QNBC tumors are more frequent in African Americans compared to TNBC tumors. To characterize this subtype further, we sought to determine the miRNA-mRNA profile in QNBC patients based on race. Both miRNA and mRNA expression data were analyzed from TCGA and validated using datasets from the METABRIC, TCGA proteomic, and survival analysis by KMPLOT. miRNA-mRNAs which include FOXA1 and MYC (mir-17/20a targets); GATA3 and CCNG2 (mir-135b targets); CDKN2A, CDK6, and B7-H3 (mir-29c targets); and RUNX3, KLF5, IL1-ß, and CTNNB1 (mir-375 targets) were correlated with basal-like and immune subtypes in QNBC patients and associated with a worse survival. Thus, QNBC tumors have an altered gene signature implicated in racial disparity and poor survival.

Obesity Influences the Expression of Key Immunomodulators in Normal Human Breast Tissue, Basal-like Breast Cancer Patients, and Cell Lines.

Among the different components of the breast cancer microenvironment are adipocytes, which are mainly composed of differentiated adipocytes and adipose progenitors. The role of obesity in tumor progression has become a key topic in clinical studies, but the mechanics of this are still misunderstood. There is significant evidence of serum amyloid (SAA1), an acute-phase protein, being heavily expressed in inflamed, septic conditions. VTCN1 and VSIR, members of the immunoglobulin family, are key players in T-cell regulation. The present study investigates the differentially expressed genes caused by adipose-conditioned media on the novel triple-negative breast cancer cell lines MDA MB 231 and MDA MB 468. RNA sequencing of adipocyte-conditioned media (ACM)-treated MDA MB 231 and MDA MB 468 cells were analyzed and compared using the gene sequencing enrichment analysis database (GSEA). GSEA was also done on microarray data from obese, non-tumorous breast tissue patients (GSE:33526) to show significantly upregulated immunomodulators. Obesity was also shown to influence gene expression related to immune sensing and evasion in a dataset analysis of basal-like obese patients (GSE:79858). We showed obesity significantly upregulated immunomodulators related to immune suppression in non-tumorous, basal-like patients, as well as in novel basal-like TNBC cell lines.

Development of OX40 agonists for canine cancer immunotherapy.

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

African Ancestry-Associated Gene Expression Profiles in Triple-Negative Breast Cancer Underlie Altered Tumor Biology and Clinical Outcome in Women of African Descent.

Women of sub-Saharan African descent have disproportionately higher incidence of triple-negative breast cancer (TNBC) and TNBC-specific mortality across all populations. Population studies show racial differences in TNBC biology, including higher prevalence of basal-like and quadruple-negative subtypes in African Americans (AA). However, previous investigations relied on self-reported race (SRR) of primarily U.S. populations. Due to heterogeneous genetic admixture and biological consequences of social determinants, the true association of African ancestry with TNBC biology is unclear. To address this, we conducted RNA sequencing on an international cohort of AAs, as well as West and East Africans with TNBC. Using comprehensive genetic ancestry estimation in this African-enriched cohort, we found expression of 613 genes associated with African ancestry and 2,000+ associated with regional African ancestry. A subset of African-associated genes also showed differences in normal breast tissue. Pathway enrichment and deconvolution of tumor cellular composition revealed that tumor-associated immunologic profiles are distinct in patients of African descent. SIGNIFICANCE: Our comprehensive ancestry quantification process revealed that ancestry-associated gene expression profiles in TNBC include population-level distinctions in immunologic landscapes. These differences may explain some differences in race-group clinical outcomes. This study shows the first definitive link between African ancestry and the TNBC immunologic landscape, from an African-enriched international multiethnic cohort. See related commentary by Hamilton et al., p. 2496. This article is highlighted in the In This Issue feature, p. 2483.

Characterization of Canine Adenovirus Type 2 Virus Infection Pattern in Canine and Human Cell Lines.

Canine adenovirus type 2 (CAV2) is a nonhuman adenovirus with a known ability to infect human and canine cells. The cell surface receptors involved in CAV2 transduction are still unknown. Identification of these would provide valuable information to develop enhanced gene delivery tools and better understand CAV2 biology. CAV2 is erroneously grouped with Ad5 based on the knowledge that CAV2 may transduce using CAR. Therefore, we have evaluated CAV2 and Ad5 (CAV2GFP, Ad5G/L) infection patterns in various canine and human cell lines to determine their different tropisms. Our research demonstrates that CAV2 can successfully infect cells that Ad5 does not infect, and CAV2 infections do not correlate with CAR expression. CAV2 can infect cells that have a low or minimal expression of CAR. Our data suggest that CAV2 transduction is not dependent on the CAR receptor, and thus, it is crucial to find novel CAV2 receptors.

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients.

Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.

Camptothecin Induces PD-L1 and Immunomodulatory Cytokines in Colon Cancer Cells.

: Background: Immunotherapy has changed the options for the treatment of various cancer types, but not colon cancer. Current checkpoint blockade approaches are ineffective in a large proportion of colon cancer cases, necessitating studies to elucidate its mechanisms and to identify new targets and strategies against it. Methods: Here, we examined Programmed Death-Ligand 1(PD-L1), cytokine and receptor responses of colon cancer cells exposed to camptothecin (CPT), a clinically used topoisomerase inhibitor. Colon cancer cells were treated with CPT at concentrations of up to 10 µM, and the expressions of PD-L1 and immunoregulatory cytokine genes and receptors were analyzed. Results: PD-L1, a current immunotherapy target for various cancers, was shown to be upregulated in colon cancer cells independent of the cellular p53 status. In metastasis-derived SW620 cells, CPT most extensively upregulated cytokines with T-cell attraction or growth factor functions. Of those modulated genes, SPP1, IL1RN, IL1A, TNFSF13B, OSM, and CSF3 had the most clinical relevance, as their high expression was associated with poor cancer patient overall survival. Conclusions: These findings highlight the need to examine, in preclinical and clinical situations, the potential benefits of combining topoisomerase inhibitors with immune-checkpoint inhibitors.

Quadruple Negative Breast Cancers (QNBC) Demonstrate Subtype Consistency among Primary and Recurrent or Metastatic Breast Cancer.

PURPOSE: Despite the availability of current standards of care treatments for triple negative breast cancer (TNBC), many patients still die from this disease. Quadruple negative tumors, which are TNBC tumors that lack androgen receptor (AR), represent a more aggressive subtype of TNBC; however, the molecular features are not well understood. METHODS: Immunohistochemistry of estrogen receptor (ER), progesterone receptor (PR), HER2, and AR was determined in 244 primary and 630 recurrent/metastatic site biopsies. Expression was correlated with a panel of 25 cancer-related genes and proteins by IHC and in situ hybridization (ISH). RESULTS: We observed that 80.2% (65 of 81) of primary TNBC tumors and 75.7% (159 of 210) of recurrent/metastatic TNBC tumors are QNBC. Bivariate fit analysis demonstrated that QNBC (n = 224) significantly (P < .03) correlated with younger aged patients at initial biopsy compared to AR positive TNBC patients (n = 51). In paired primary tissue samples and primary to recurrent/metastatic samples, at least 70% Luminal, HER2 enriched, and QNBC subtype did not change molecular profile. But, TNBC seems to be the "unstable" subtype. Within the total cohort, discordance in molecular profiles was identified in both synchronous (20%) and asynchronous (21%) intra-individual analyses. Irrespective of sample type, (Synchronous or Asynchronous), QNBC demonstrated higher concordant than TNBC. IHC and ISH results of the cancer related genes, demonstrated that gene/protein expression differ by molecular profile: TNBC (HR-/HER2-, AR+) and QNBC (HR-/HER2-, AR-). IHC in metastatic tumors, showed that the percentage of tumors positive of EGFR were higher, while PTEN and TLE3 were lower in QNBC compared to TNBC. CONCLUSION: Standard treatment of Breast Cancer (BC) relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our analyses suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant BC subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies.

Phage Ligands for Identification of Mesenchymal-Like Breast Cancer Cells and Cancer-Associated Fibroblasts.

Epithelial to mesenchymal transition (EMT) is believed to be crucial for primary tumors to escape their original residence and invade and metastasize. To properly define EMT, there is a need for ligands that can identify this phenomenon in tumor tissue and invivo. A phage-display selection screening was performed to select novel binding phage peptides for identification of EMT in breast cancer. Epithelial breast cancer cell line, MCF-7 was transformed to mesenchymal phenotype by TGF-ß treatment and was used for selection. Breast fibroblasts were used for subtractive depletion and breast cancer metastatic cell lines MDA-MB-231, T47D-shNMI were used for specificity assay. The binding peptides were identified, and their binding capacities were confirmed by phage capture assay, phage-based ELISA, immunofluorescence microscopy. The phage peptide bearing the 7-amino acid sequence, LGLRGSL, demonstrated selective binding to EMT phenotypic cells (MCF-7/TGF-ß and MDA-MB-231) as compared to epithelial subtype, MCF-7, T47D and breast fibroblasts (Hs578T). The selected phage was also able to identify metastatic breast cancer tumor in breast cancer tissue microarray (TMA). These studies suggest that the selected phage peptide LGLRGSL identified by phage-display library, showed significant ability to bind to mesenchymal-like breast cancer cells/ tissues and can serve as a novel probe/ligand for metastatic breast cancer diagnostic and imaging.

Neuroligin 4X overexpression in human breast cancer is associated with poor relapse-free survival.

The molecular mechanisms involved in breast cancer progression and metastasis still remain unclear to date. It is a heterogeneous disease featuring several different phenotypes with consistently different biological characteristics. Neuroligins are neural cell adhesion molecules that have been implicated in heterotopic cell adhesion. In humans, alterations in neuroligin genes are implicated in autism and other cognitive diseases. Until recently, neuroligins have been shown to be abundantly expressed in blood vessels and also play a role implicated in the growth of glioma cells. Here we report increased expression of neuroligin 4X (NLGN4X) in breast cancer. We found NLGN4X was abundantly expressed in breast cancer tissues. NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) was analyzed. Correlation between NLGN4X levels and clinicopathologic parameters were analyzed within Oncomine datasets. Evaluation of these bioinfomatic datasets results revealed that NLGN4X expression was higher in triple negative breast cancer cells, particularly the basal subtype and tissues versus non-triple-negative sets. Its level was also observed to be higher in metastatic tissues. RT-PCR, flow cytometry and immunofluorescence study of MDA-MB-231 and MCF-7 breast cancer cells validated that NLGN4X was increased in MDA-MB-231. Knockdown of NLGN4X expression by siRNA decreased cell proliferation and migration significantly in MDA-MB-231 breast cancer cells. NLGN4X knockdown in MDA-MB-231 cells resulted in induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. High NLGN4X expression highly correlated with decrease in relapse free-survival in TNBC. NLGN4X might represent novel biomarkers and therapeutic targets for breast cancer. Inhibition of NLGN4X may be a new target for the prevention and treatment of breast cancer.

Regulation of intracellular trafficking and secretion of adiponectin by myosin II.

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB.

Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins.

Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox). Previously, we have shown that isolated pVIII major coat proteins of the fd-tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N-terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity toward breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed). Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7), three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05) in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity toward a doxorubicin-insensitive pancreatic cancer line (PANC-1) showed significant increases in toxicity (2-fold; p < 0.05). Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.

Selection of pancreatic cancer cell-binding landscape phages and their use in development of anticancer nanomedicines.

It is hypothesized that the use of targeted drug delivery systems can significantly improve the therapeutic index of small molecule chemotherapies by enhancing accumulation of the drugs at the site of disease. Phage display offers a high-throughput approach for selection of the targeting ligands. We have successfully isolated phage fusion proteins selective and specific for PANC-1 pancreatic cancer cells. Doxorubicin liposomes (Lipodox) modified with tumor-specific phage fusion proteins enhanced doxorubicin uptake specifically in PANC-1 cells as compared with unmodified Lipodox and also compared with normal breast epithelial cells. Phage protein-targeted Lipodox substantially increased the concentration of doxorubicin in the nuclei of PANC-1 cells in spite of P-glycoprotein-mediated drug efflux. The in vitro cytotoxic activity obtained with pancreatic cell-targeted Lipodox was greater than that of unmodified Lipodox. We present a novel and straightforward method for preparing pancreatic tumor-targeted nanomedicines by anchoring pancreatic cancer-specific phage proteins within the liposome bilayer.

Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Landscape phages and their fusion proteins targeted to breast cancer cells.

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein.

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG(2k)-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.

Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors.

One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.