Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Deregulation of AKT-mTOR Signaling Contributes to Chemoradiation Resistance in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (LUSC) accounts for one of three of non-small cell lung carcinoma (NSCLC) and 30% of LUSC patients present with locally advanced, unresectable/medically inoperable disease, who are commonly treated with definitive chemoradiation. However, disease relapse in the radiation fields occurs in one of three cases. We aim to explore the underlying molecular mechanisms of chemoradiation resistance of LUSC. Patient-derived xenograft (PDX) models of LUSC were established in immunodeficient mice, followed by treatment with cisplatin in combination with clinically relevant courses of ionizing radiation (20, 30, and 40 Gy). The recurrent tumors were extracted for functional proteomics using reverse phase protein analysis (RPPA). We found that phospho-AKT-S473, phospho-AKT-T308, phospho-S6-S235/6, and phospho-GSK3ß-S9 were upregulated in the chemoradiation-resistant 20 Gy + cisplatin and 40 Gy + cisplatin tumors compared with those in the control tumors. Ingenuity pathway analysis of the RPPA data revealed that AKT-mTOR signaling was the most activated signaling pathway in the chemoradiation-resistant tumors. Similarly, elevated AKT-mTOR signaling was observed in stable 40 Gy and 60 Gy resistant HARA cell lines compared with the parental cell line. Accordingly, pharmacologic inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. In conclusion, using chemoradiation-resistant PDX models coupled with RPPA proteomics analysis, we revealed that deregulation of AKT-mTOR signaling may contribute to the chemoradiation resistance of LUSC. IMPLICATIONS: Clonal selection of subpopulations with high AKT-mTOR signaling in heterogeneous tumors may contribute to relapse of LUSC after chemoradiation. mTOR kinase inhibitors may be promising radiosensitizing agents in upfront treatment to prevent acquired resistance.

Altered Gemcitabine and Nab-paclitaxel Scheduling Improves Therapeutic Efficacy Compared with Standard Concurrent Treatment in Preclinical Models of Pancreatic Cancer.

PURPOSE: Concurrent gemcitabine and nab-paclitaxel treatment is one of the preferred chemotherapy regimens for metastatic and locally advanced pancreatic ductal adenocarcinoma (PDAC). Previous studies demonstrate that caveolin-1 (Cav-1) expression is critical for nab-paclitaxel uptake into tumors and correlates with response. Gemcitabine increases nab-paclitaxel uptake by increasing Cav-1 expression. Thus, we hypothesized that pretreatment with gemcitabine would further enhance the sensitivity of PDAC to nab-paclitaxel by increasing Cav-1 expression and nab-paclitaxel uptake. EXPERIMENTAL DESIGN: We investigated the sensitivity of different gemcitabine and nab-paclitaxel treatment regimens in a panel of PDAC cell lines and orthotopic xenograft models. The sensitivity of different treatment regimens was compared with the standard concurrent treatment. RESULTS: Pretreatment with gemcitabine before nab-paclitaxel increased Cav-1 and albumin uptake and significantly decreased proliferation and clonogenicity compared with concurrent treatment, which correlated with increased levels of apoptosis. Cav-1 silencing reduced the uptake of albumin, and therapeutic advantage was observed when cells were pretreated with gemcitabine prior to nab-paclitaxel. In addition, we observed that pretreatment with gemcitabine resulted in partial synchronization of cells in the G2-M-phase at the time of nab-paclitaxel treatment, providing another mechanism for the benefit of altered scheduling. In heterotopic and orthotopic xenograft models, the altered schedule of gemcitabine prior to nab-paclitaxel significantly delayed tumor growth compared with concurrent delivery without added toxicity. CONCLUSIONS: Pretreatment with gemcitabine significantly increased nab-paclitaxel uptake and correlated with an increased treatment efficacy and survival benefit in preclinical models, compared with standard concurrent treatment. These results justify preclinical and clinical testing of this altered scheduling combination.

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs.

Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

Polyanhydride nanovaccine against swine influenza virus in pigs.

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8αα+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a leading cause of economic burden to the pork industry worldwide. The routinely used modified live PRRS virus vaccine (PRRS-MLV) induces clinical protection, but it has safety concerns. Therefore, in an attempt to develop a safe and protective inactivated PRRSV vaccine, we generated PRRS-virus-like-particles (PRRS-VLPs) containing the viral surface proteins GP5-GP4-GP3-GP2a-M or GP5-M using a novel baculovirus expression system. Our in vitro results indicated that the desired PRRSV proteins were incorporated in both the VLPs preparations based on their reactivity in immunogold electron microscopy and ELISA. To boost their immunogenicity in pigs, we entrapped the PRRS-VLPs in PLGA nanoparticles and coadministered them intranasally with a potent adjuvant. We then evaluated their efficacy in pigs against a viral challenge using a virulent heterologous field isolate. Our results indicated that PRRS-VLPs induced an anamnestic immune response, since we observed boosted IgG and IFN-γ production in vaccinated and virus-challenged animals, but not during the pre-challenge period. Importantly, a two-log reduction in the lung viral load was detected in PRRS-VLP-vaccinated animals. In conclusion, we generated PRRS-VLPs containing up to five viral surface proteins and demonstrated their immunogenicity in pigs, but further studies are required to improve its immunogenicity and efficacy as a vaccine candidate.

Comparative analysis of routes of immunization of a live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in a heterologous virus challenge study.

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), which infects primarily the respiratory tract of pigs. Thus intranasal (IN) delivery of a potent vaccine-adjuvant formulation is promising. In this study, PRRS-MLV (VR2332) was coadministered ± an adjuvant Mycobacterium vaccae whole cell lysate or CpG ODN through intramuscular (IM) or IN route as a mist, and challenged with a heterologous PRRSV 1-4-4 IN at 42 days post-vaccination (dpv). At 14 and 26 dpv, vaccine viral RNA copies were one log greater in the plasma of PRRS-MLV IM compared to IN vaccinated pigs, and the infectious replicating vaccine virus was detected only in the IM group. In PRRS-MLV ± adjuvant IM vaccinated pigs, reduced viral RNA load and absence of the replicating challenged virus was observed at 7, 10 and 14 days post-challenge (dpc). At 14 dpc, in BAL fluid ≥ 5 log viral RNA copies were detected in all the pig groups, but the replicating challenged virus was undetectable only in IM groups. Immunologically, virus neutralizing antibody titers in the plasma of IM (but not IN) vaccine groups was ≥ 8 against the vaccine and challenged viruses. At 26 dpv, PRRS-MLV IM (without adjuvant) received pigs had significantly increased population of CD4 and CD8 T cells in PBMC. At 14 dpc, relatively increased population of IFN-γ(+) total lymphocytes, NK, CD4, CD8 and γδ T cells were observed in the MLV-IM group. In conclusion, PRRS-MLV IM vaccination induced the virus specific T cell response in pigs, but still it is required to improve its cross-protective efficacy.

Mutations in a Highly Conserved Motif of nsp1ß Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (nsp1ß) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1ß to suppress interferon beta (IFN-ß) activation and also impaired nsp1ß's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1ß mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1ß mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1ß function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE: PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1ß was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1ß function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.

Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions.

Porcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.