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PurposeThe study aims to examine the customers' revisit intention toward the green restaurants after service failure based upon service failure attributions. The study further intends to investigate the moderating effect of green self-identity on customers' post-service failure behavioral intentions.Design/methodology/approachA self-administered questionnaire was distributed to 327 participants who had experienced service failure while dining in green restaurants. The study draws upon the prevailing literature to examine the relationship among the constructs using structural equation modeling (SEM).FindingsThe findings of the study have confirmed that service failure has an adverse effect on customers' revisit intention toward the green restaurants. However, customers with green self-identity appear less anxious about service failure as findings indicate customers revisit green restaurant even after service failure.Practical implicationsThe study provides a clear indication to the managers of the green restaurants that a better understanding of service failure attributions may facilitate in preventing service failure in a prompt and reasonable manner. It will not only contribute to building the brand reputation, but also ensure that customers stay with the brand for a longer duration.Originality/valueThe study is unique in a way that it is the first of its type to establish a relationship between service failure attributions and customer satisfaction in the emerging South Asian market, such as India in the context of green restaurants. Besides, this is the only study to use green self-identity as a moderator between the relationships of customer satisfaction and revisit intention.

Table 4 Excel Document

Supplemental Figures 1-8, Supplemental Tables 1-3

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Legends for Supplementary Figures 1-8 and Supplementary Tables 1-5

Table 5 Excel Document

Supplemental Figure 1. Rac1 is a driver of ccRCC. (A) Rac1 mRNA expression in normal kidney and ccRCC patients from Jones Renal Cohort. (B) Rac1 DNA copy number analysis of normal blood, normal kidney, and kidney tumors from the KIRC TCGA dataset. (C) Rac isoform expression analysis in normal kidney and kidney tumors from the TCGA KIRC dataset. (D) PREX1, VAV1, TIAM1, and TRIO mRNA expression in normal kidney and kidney tumors from the TCGA KIRC dataset. (E) Rac1 mRNA expression by clinical stage from the TCGA KIRC dataset. (F) Boyden chamber cellular migration assay of 786-O transfected with siNSC or siRac1 oligonucleotides. (G) Cellular proliferation assay of 786-O transfected with siNSC or siRac1 oligonucleotides.

Figure S1 shows Rac1 expression analysis of Normal Colon and Colon Adenocarcinoma patients obtained from the TCGA COAD dataset using Oncomine. Data is represented as Log2 Median centered ratio.

Supplemental Figure 2. HACE1 overexpression reduces activated Rac1 and attenuates ccRCC growth and survival. (A) Rac1 and HACE1 mRNA expression correlation from the KIRC TCGA Dataset (B) Rac1-GTP Pull-down assay of empty vector or HACE1 overexpressing 786-O cells. (C) Migration assay on empty vector or HACE1 overexpressing 786-O cells. (D) Cellular proliferation assay of empty vector or HACE1 overexpressing 786-O cells. (E) Soft agar assay results of empty vector or HACE1 overexpressing 786-O cells.

Resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. Novel strategies to circumvent resistance are critical to prolonging patient survival. Rac1b, a constitutively activated isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NF-κB signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NF-κB activity. Rac1b knockdown reduced cellular proliferation and reduced NF-κB activity. Surprisingly, Rac1b expression and NF-κB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NF-κB signaling. Knockdown of Rac1b or Rac inhibition increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NF-κB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Although Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared with single agents alone. We provide the first evidence that Rac1b expression confers resistance to chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NF-κB signaling, providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo. Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.

Supplementary Figures S1-S3; Supplementary Materials and Methods; Supplementary Figure Legend; Supplementary Table S1

Supplemental Figure 3. In vitro and In Vivo characterization of GYS32661. (A) Cellular proliferation assay of 786-O cells treated with vehicle, 2.5, and 5 μM GYS32661. (B) Plasma and tumor concentrations of GYS32661 at 0.25, 1, 4, and 8 hour time points.

MUC4, a transmembrane mucin, is aberrantly expressed in pancreatic adenocarcinomas while remaining undetectable in the normal pancreas. Recent studies have shown that the expression of MUC4 is associated with the progression of pancreatic cancer and is inversely correlated with the prognosis of pancreatic cancer patients. In the present study, we have examined the phenotypic and molecular consequences of MUC4 silencing with an aim of establishing the mechanistic basis for its observed role in the pathogenesis of pancreatic cancer. The silencing of MUC4 expression was achieved by stable expression of a MUC4-specific short hairpin RNA in CD18/HPAF, a highly metastatic pancreatic adenocarcinoma cell line. A significant decrease in MUC4 expression was detected in MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with the parental and scrambled short interfering RNA–transfected (CD18/HPAF-Scr) control cells by immunoblot analysis and immunofluorescence confocal microscopy. Consistent with our previous observation, inhibition of MUC4 expression restrained the pancreatic tumor cell growth and metastasis as shown in an orthotopic mouse model. Our in vitro studies revealed that MUC4-associated increase in tumor cell growth resulted from both the enhanced proliferation and reduced cell death. Furthermore, MUC4 expression was also associated with significantly increased invasiveness (P ≤ 0.05) and changes in actin organization. The presence of MUC4 on the cell surface was shown to interfere with the tumor cell-extracellular matrix interactions, in part, by inhibiting the integrin-mediated cell adhesion. An altered expression of growth- and metastasis-associated genes (LI-cadherin, CEACAM6, RAC1, AnnexinA1, thrombomodulin, epiregulin, S100A4, TP53, TP53BP, caspase-2, caspase-3, caspase-7, plakoglobin, and neuregulin-2) was also observed as a consequence of the silencing of MUC4. In conclusion, our study provides experimental evidence that supports the functional significance of MUC4 in pancreatic cancer progression and indicates a novel role for MUC4 in cancer cell signaling. (Mol Cancer Res 2007;5(4):309–20)

PDF file - 135K, Figure S2. Efficiency of PLOD2 knockdown in MDA-MB-435 cells.

PDF file - 98K, Figure S1. HIF-1α is required for PLOD induction under hypoxia in mouse embryo fibroblasts.

Supplementary Figure S1. Cell proliferation of U87-MG subclones in vitro. 5,000 cells were seeded and cultured under 20% or 1% O2. Viable cell number was counted by trypan blue staining on days 2, 3, and 4, and normalized to the starting cell number (mean + SEM, n = 3). Supplementary Figure S2. Analysis of orthotopic tumors after injection of 1 x 104 cells into the caudate-putamen. (A) Tumor volume was determined by histological analysis 5 weeks after stereotactic injection of U87-MG parental or subclone cells into the brains of SCID mice (bar = mean, n = 7); *p < 0.05 vs shNT. (B) Depth of invasion was determined by histological analysis (mean + SEM, n = 5-7). (C) Anti-CD105 immunohistochemistry was performed and blood vessel density in tumor sections was determined (mean + SEM, n = 10-14). (D) Anti-Ki-67 immunohistochemistry was performed and stained cells were counted (mean + SEM, n = 5-7). (E) Immunohistochemistry was performed to detect active caspase-3 and stained cells were counted (mean + SEM, n = 5-7). Supplementary Figure S3. HIF-2α mRNA levels were determined by RT-qPCR (mean + SEM, n = 3) in parental, shScr, shCBS#07, and shCBS#61 subclones cultured at 20% O2.

Table S1 Shows Rac1 Immunohistochemistry scores for normal colon and colon cancer by stage

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.

PurposeThe current study has drawn attention to investigating the impact of social media influencers’ (SMIs) authenticity on followers buying behavior by using followers who have an ongoing relationship with an influencer and are knowledgeable about the influencer. The study further intends to reveal the mediating effect of parasocial interaction on the relationship between SMI's authenticity and followers' purchase behavior.Design/methodology/approachThe study has analyzed data from an online survey of 458 participants (Instagram followers) using structured equation modeling (CB-SEM) to investigate the relationship among authenticity attributes, parasocial interaction and followers' purchase behavior.FindingsCB-SEM results reveal that authenticity attributes positively influence followers' buying behavior. The findings from mediation analysis specify that parasocial interaction mediates the relationship between authenticity attributes (sincerity, truthful endorsement and visibility) and buying behavior excluding expertise, uniqueness attributes.Practical implicationsThe findings of the study reinforce the need to use authentic influencers by the marketers for the brand endorsements. Further, the findings of the study can benefit marketers in implementing strategic practice of social media influencer marketing.Originality/valueThe study overcomes the limitations of preceding studies by using Instagram followers who are well-informed about SMIs and have an ongoing relationship with them. This study has uniquely combined the behavioral data from real influencer campaigns with followers' assessment of an influencer's authenticity.

PurposeAnthropogenic activities such as unsustainable consumption pattern is one of the reasons responsible for the ongoing environmental issues. Although, consumers are becoming increasingly aware and concerned about environmental problems their attitudes are not resulting in sustainable consumption behavior (SCB). Celebrity institutional entrepreneurs can engage and inspire the public at large and contribute to institutional change. Hence, this study aims to explore the potential of celebrity institutional entrepreneurship in galvanizing mainstream SCB by increasing the awareness of environmental issues and their consequences.Design/methodology/approachThis study examines the actor's influence by conducting a netnographic analysis of Leonardo DiCaprio's Instagram account. Further, qualitative interviews of account followers were also conducted to evaluate the influence of account on their awareness levels and consumption practices.FindingsOur findings indicate that account had a significant impact on consumers' environmental awareness and engagement with environmental issues. However, a partial impact was seen in case of their sustainable consumption practices. Our study concludes that celebrity institutional entrepreneurship can help in addressing the attitude-behavior gap in sustainability research.Originality/valueThis study is amongst the few studies that attempted to explore the ways to reduce the attitude-behavior gap in SCB. It examines the potential of celebrity institutional entrepreneurship to galvanize mainstream sustainable consumption. The results of this study are useful to key stakeholders (policymakers, marketers, social-environmental groups etc.) in the development of more effective strategies for sustainable development.

PurposeThe purpose of this study is to conceptualize and analyze a framework that provides greater understanding toward the impact of service recovery antecedents such as role clarity, customer service orientation, employee empowerment and employee relational behavior on customer satisfaction and customer delight in the context of quick-service restaurants (QSRs).Design/methodology/approachA self-administered questionnaire was distributed to 408 participants who had experienced service recovery efforts by leading QSRs on social media. The current paper draws upon the prevailing literature to test a series of research hypotheses through structural equation modeling.FindingsThe findings of the study have confirmed that antecedents of service recovery are good to describe customer satisfaction and customer delight in the setting of QSRs. Besides, the study provides an understanding on how monetary compensation moderates the relationship between customer delight and customer satisfaction.Practical implicationsThis study carries an understanding on how frontline employees must operate in a non-conventional and innovative way to resolve customers' issues and show commitment with truthfulness to provide excellent services to make customers feel delightful.Originality/valueThis is a unique study to understand the role of service recovery antecedents to describe customer satisfaction and customer delight in the social media environment. In addition, the results support the possibilities of implementing prompt service recovery efforts using social media.

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity in vitro and sequesters plasma TGF-β in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.

PurposeThe current study aims to investigate the various consumption motives (hedonic, gain and normative) responsible for strengthening consumers' intentions toward purchase behavior for electric vehicle (EV).Design/methodology/approachA total of 411 valid survey responses were collected using a structured questionnaire. Data were analyzed using confirmatory factor analysis and structural equation modeling to investigate the empirical fit of the hypothesized framework.FindingsThe results of structural equation modeling revealed that all three motives were positively correlated with purchase intentions for EV. Hedonic motives were found to have the strongest influence on purchase intentions. In addition, gain and normative motives were also found to be significant predictors of EV buying behavior. Further analysis revealed a positive correlation between gain, normative and hedonic motives. Moreover, personal moral standards seem to have a significant and positive impact on the positive emotions associated with buying EV.Practical implicationsThe results of current research can be useful for marketers while designing promotional strategies for all the high-involvement green products. Marketing professionals and policymakers can use these results to build effective marketing strategies for EVs and reduce greenhouse gas emissions resulting from personal vehicle use.Originality/valueTo the best of the authors' knowledge, this is the first study in the South Asian region that explores consumers' motives for EV purchase behavior. Further, this is among a few studies, which have attempted to investigate the impact of hedonic, gain and normative motives on green purchase behavior in the context of high involvement green products.

Endothelial adherens junctions (AJs) are critical for the regulation of vascular barrier integrity and undergo dis-assembly during inflammatory injury, thus causing vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial barrier following the initial injury. Here we examine the metabolic underpinnings that drive restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. We engineered a novel chemo-genetic construct (RapT) which allowed for precise temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by rapamycin during the barrier restoration phase increased regional ATP and accelerated AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 activity. Using another chemo-genetic approach to temporally control Src activity, we found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen species generated during inflammatory stimulation create pockets of regional hypoxia and allow for increased Hif1α-mediated transcription of PFKFB3, leading to sustained glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration of vascular integrity both in vitro and in vivo. In conclusion, we show that while inflammatory activation acutely compromises the endothelial barrier, inflammatory signaling also concomitantly generates a metabolic milieu in anticipation of the subsequent re-assembly of AJs and restoration of the vascular barrier.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

PurposeThis study aims at analyzing the impact of green restaurants' sustainable practices (food safety, food sustainability practices, food quality, and environmental sensitivity) on consumer satisfaction and revisit intention.Design/methodology/approachA self-administered questionnaire was used to collect data from 417 participants who had recently experienced the services of leading green restaurants. The study draws upon the prevailing literature to test a series of research hypotheses through structural equation modeling.FindingsThe findings of the study have confirmed that sustainable practices such as food safety, food sustainability practices, environmental sensitivity, and food quality significantly influence consumers' satisfaction and revisit intentions. The willingness to pay has been found as a sensitive issue as it moderates the relationship between consumer satisfaction and revisit intention.Practical implicationsThis study provides important insights for the businesses operating in foodservice industry. The study suggests important strategies to the restaurant business owners for improving their consumers' satisfaction and revisit intentions. These strategies may help foodservice businesses in building strong brand reputation and a competitive edge over others.Originality/valueThis study contributes to the food service literature by examining the restaurants' environmental legitimacy (food safety, food sustainability practices, food quality, and environmental sensitivity) from consumers' perspective. Moreover, the current study also fills the gap in literature by expanding the knowledge of consumers' pro-environmental behavior in the context of developing nations. Besides, to the best of the authors' knowledge, this is among very few studies, which have emphasized on analyzing consumer satisfaction and revisit intention based on their experience in a green restaurant on particular sustainable parameters.

Immunotherapeutics that aid in boosting natural immune defenses against cancers have revolutionized cancer treatment. Previously, we reported a novel heterodimeric bifunctional fusion molecule, HCW9218, designed using soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex which exhibited both immune cell stimulatory and TGF-β neutralizing properties. Herein, we showed in two different syngeneic murine tumor models (B16F10, 4T1) that subcutaneous treatment with HCW9218 induces a proliferative burst of CD8+ T cells and NK cells in blood and a subsequent infiltration of these cells into established tumors. In vivo imaging of 4T1 tumor-bearing mice after treatment showed that HCW9218 was present both in lymph nodes and established tumors up to 24hr following treatment. Comprehensive analysis of tumor infiltrating lymphocytes (TILs) showed that HCW9218 mediated antitumor activity by expanding TCF+TIM3− ‘progenitor exhausted’ (Tpex) CD8+ T cells in tumors. Sphingosine-1-phosphate receptor blockade resulted in decreased tumor infiltration of CD8+ Tpex in B16F10 and 4T1 tumor-bearing mice indicating that these cells originate from tumor draining lymph nodes (TdLN). Increased ‘terminally exhausted‘ TCF-1−TIM3+ (Tex) CD8+ TILs were also observed in tumors of HCW9218-treated mice indicating increased antitumor activity. Tumor transplantation experiments further confirmed the mechanism of HCW9218 antitumor activity by increasing influx of CD45.1+ CD8+ T cells into transplanted tumors from CD45.2+ mice. Additionally, HCW9218 enhanced the therapeutic efficacy of PD-L1 treatment by increasing the infiltration of activated/memory CD8+ T cells into B16F10 tumors in mice leading to significant reduction in tumor volume. Collectively, the results of this study demonstrated that HCW9218 treatment of mice bearing solid tumors resulted in modulating the TdLN immune landscape and invigorating T cells for enhanced checkpoint blockade therapy. HCW9218 are currently in two clinical trials (clinicaltrials.org: NCT05322408, NCT05304936) against chemo-resistant/refractory solid tumors. Citation Format: Varghese George, Pallavi Chaturvedi, Niraj Shrestha, Leah Kanakraj, Crystal Gilkes, Nicole Encalada, Meng Wang, Xiaoyun Zhu, Bai Liu, Peter Rhode, Hing C. Wong. Bifunctional immunotherapeutic HCW9218 facilitates recruitment of immune cells from tumor draining lymph nodes to promote antitumor activity and enhance checkpoint blockade efficacy in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4441.

Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1α expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1α and HIF-2α protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.

Purpose The purpose of this study is to investigate the influence of environmental concern, perceived value, personal norms and willingness to pay on generation Z’s purchase intention for recycled clothing. Design/methodology/approach The data were collected from five Indian universities. A total of 497 usable responses were analyzed. Confirmatory factor analysis was used for examining the validity and reliability of the scales. Further, the structural equation modeling was used to assess the relationship among the constructs. Findings Findings suggested that willingness to pay, environmental concern, perceived value and personal norms influence generation Z’s purchase intention for recycled clothing. Willingness to pay, environmental concern and perceived value were major predictors of purchase intention for recycled clothing. Practical implications This study holds much importance to the marketers of recycled clothing as it provides useful insights for formulating the appropriate promotional strategies. The study also contributes to the consumer behavior literature by addressing the existing research gap. Originality/value Most of the studies existing in this area have focused on the manufacturing side only except few which explored the consumption side of recycled clothing. Hence, the current study is an attempt to fill this research gap.

Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-β (TGF-β) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.

In this article, an approach has been introduced for detecting cancerous cells. Image processing techniques have been used, based on cancer cell area using CNNs. A very intriguing aspect of this experiment was that from a very small image dataset, a large number of images were generated using information augmentation which was then taken as the training set data. The suggested scheme detects cancer behaviors through a convolutional neural network in images of celled samples. Previously, the same attempts failed to stay away from the database dependencies, which were somewhat proportional to the number of images in datasets, so we used a method called data augmentation on smaller sets of images. The scheme preprocesses the input image by grayscale, binarization, inversion, median filtering, and flood-fill procedures. Depending on the sort of feature to be identified, the preprocessed image is then cancerous cell detected. This methodology was used for several sets of pictures, and the system was optimized with the feedback from those tests. For independent cancer cell detection with narrower datasets, the suggested technique can be efficiently used, which will greatly accelerate the study of cancer and open greater dimensions.

Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.

Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo. Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.

Chemotherapeutic agents not only cause apoptosis and necrosis of cancer cells but also induce cellular senescence (TIS), resulting in a persistent cell population that re-enters the cell cycle with stemness characteristics via the senescence-associated secretory phenotype. These senescent cancer cells, therefore, seed disease relapses if they are not effectively eliminated. TIS cancer cells lack HLA expression and cannot be targeted by T cells. Herein, we show that chemotherapy-induced senescent B16F10 cells express the NK ligands Rae1e and Ulbp1, and are effectively killed by mouse NK cells in vitro. Docetaxel treatment also induced senescent B16F10 cells in vivo. We have previously shown that the TGF-β trap/IL-15 fusion protein, aka HCW9218, effectively activates NK cells in vivo. Therefore, we treated B16F10 tumor bearing mice with docetaxel and then a single dose of HCW9218 in combination with the ADCC antibody TA99. This treatment regimen exhibited potent antitumor activity compared to docetaxel alone. End point analysis showed that HCW9218-treated tumors have lower mitotic activity, reduced expression of senescence markers, and increased frequencies of infiltrating CD8 T cells. We further showed in this model that HCW9218 treatment followed by anti-PD-L1 therapy was more effective than anti-PD-L1 therapy followed by HCW9218. These results suggest that the optimal sequence of cancer treatment is: (1) standard-of-care chemotherapy to control tumor burden and induce cellular senescence, (2) immunotherapy for removal of senescent cells and enhanced tumor infiltration of CD8+ T cells and (3) checkpoint blockade to maintain infiltrated T cell effector function to enhance therapeutic responses and treatment durability.

Interleukin-15 (IL-15) is a promising cytokine for cancer therapy. However, it has shown limited antitumor efficacy as monotherapy in clinical trials to date. Removal of immunosuppression of the tumor microenvironment is considered a promising approach to enhance IL-15-mediated immune responses. TGF-β is a key component for creating an immunosuppressive tumor microenvironment. Therefore, we constructed a novel bifunctional fusion protein complex, designated HCW9218, comprising a soluble fusion of two TGFβRII, human tissue factor, and human IL-15, and a second soluble fusion of two TGFβRII and a sushi domain of IL-15Rα. HCW9218 activates IL-15R signaling and the dimeric TGFβRII functions as “trap” for all the three TGF-β isoforms. In healthy and tumor-bearing C57/BL6 mice, subcutaneously administrated HCW9218 was well tolerated with a long serum half-life and was able to stimulate and induce proliferation of CD8+ T cells and NK cells. Splenocytes from HCW9218-treated mice showed enhanced metabolic activity and cytotoxicity against Yac-1 target cells compared to untreated mouse splenocytes. In the syngeneic B16F10 melanoma mouse model, a single-dose of HCW9218 exhibited strong antitumor activity in combination with chemotherapy and TA99 antibody. HCW9218 also significantly increased immune cell infiltration into tumors. Mutagenesis studies demonstrated that both the TGFβRII and IL-15/IL-15Rα domains are essential for antitumor efficacy of HCW9218. Collectively, our preclinical data demonstrate that HCW9218 elicits potent immune responses with a remarkable safety profile and serves as a novel immunotherapeutic against cancer alone or in combination with therapeutic antibodies.

We have constructed two heterodimeric multi-cytokine fusions, HCW9201 and HCW9206, to support a “Kick and Expand” approach to activate and induce proliferation of purified NK cells for adoptive cell therapy (ACT). HCW9201 is a heterodimeric fusion protein complex comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-18 and IL-12. HCW9206 is heterodimeric fusion protein comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-7, and IL-21. When purified human NK cells from peripheral blood were activated with HCW9201 for three hours and then incubated with HCW9206 in combination with a capture antibody, the HCW9201-activated NK cells expanded approximately 100–300 folds within 14 days. The expanded NK cells exhibit a cytokine-induced memory-like (CIML) phenotype with a high metabolic rate and respiratory capacity, remarkable anti-tumor activity, and persistence when they were adoptively transferred into NSG mice. They also retained their heightened responsiveness when re-stimulated with tumor targets. Cytokine dependent epigenetic demethylation imprints of the Ifng promoter region were also observed for at least 10 days after the NK cells were adoptively transferred to NSG mice. In conclusion, a simple, scalable, non-feeder-cell-based “Kick and Expand” process was developed to support the generation of large numbers of CIML NK cells for multiple rounds of ACT using peripheral blood NK cell. We also provide data to show that these CIML NK cells are an excellent source for generation of CAR-NK cells.

Resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. Novel strategies to circumvent resistance are critical to prolonging patient survival. Rac1b, a constitutively activated isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NF-κB signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NF-κB activity. Rac1b knockdown reduced cellular proliferation and reduced NF-κB activity. Surprisingly, Rac1b expression and NF-κB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NF-κB signaling. Knockdown of Rac1b or Rac inhibition increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NF-κB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Although Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared with single agents alone. We provide the first evidence that Rac1b expression confers resistance to chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NF-κB signaling, providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.

Electroencephalography is the recording of electrical activity along the scalp of a human brain. It is most often used for clinical purposes and for brain-computer interface. The Electroencephalogram signals which record the neural activity are contaminated with Eye Blink artifacts a lot which leads to problems in brain Computer Interface systems and also in clinical diagnosis. To analyze these signals it is necessary that the EEG recording should be artifact free. In this paper, we study about the eye blink artifacts which make the electroencephalogram noisy and then we detect the presence of these eye blink artifacts and localize and visualize them using Spectrogram and Continuous Wavelet Transform.

Targeted therapy against triple-negative breast cancers, which lack expression of the estrogen, progesterone, and HER2 receptors, is not available and the overall response to cytotoxic chemotherapy is poor. One of the molecular hallmarks of triple-negative breast cancers is increased expression of genes that are transcriptionally activated by hypoxia-inducible factors (HIFs), which are implicated in many critical aspects of cancer progression including metabolism, angiogenesis, invasion, metastasis, and stem cell maintenance. Ganetespib is a second-generation inhibitor of heat shock protein 90 (HSP90), a molecular chaperone that is essential for the stability and function of multiple client proteins in cancer cells including HIF-1α. In this study, human MDA-MB-231 and MDA-MB-435 triple-negative breast cancer cells were injected into the mammary fat pad of immunodeficient mice that received weekly intravenous injections of ganetespib or vehicle following the development of palpable tumors. Ganetespib treatment markedly impaired primary tumor growth and vascularization, and eliminated local tissue invasion and distant metastasis to regional lymph nodes and lungs. Ganetespib treatment also significantly reduced the number of Aldefluor-positive cancer stem cells in the primary tumor. Primary tumors of ganetespib-treated mice had significantly reduced levels of HIF-1α (but not HIF-2α) protein and of HIF-1 target gene mRNAs encoding proteins that play key roles in angiogenesis, metabolism, invasion, and metastasis, thereby providing a molecular basis for observed effects of the drug on the growth and metastasis of triple-negative breast cancer.Triple-negative breast cancers (TNBCs) respond poorly to available chemotherapy. TNBCs overexpress genes regulated by hypoxia-inducible factors (HIFs). Ganetespib induces degradation of HSP90 client proteins, including HIF-1α. Ganetespib inhibited TNBC orthotopic tumor growth, invasion, and metastasis. Ganetespib inhibited expression of HIF-1 target genes involved in TNBC progression.

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.

Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyperglycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-kB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors. publisher: Elsevier articletitle: Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy journaltitle: Cancer Cell articlelink: http://dx.doi.org/10.1016/j.ccell.2016.10.006 associatedlink: http://dx.doi.org/10.1016/j.ccell.2016.11.007 content_type: article copyright: © 2016 Elsevier Inc. ispartof: Cancer Cell vol:30 issue:6 pages:968-985 ispartof: location:United States status: published

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.

Acquired resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. New strategies to circumvent this problem are critical to prolonging patient survival. Previous studies have shown that Rac1b, a constitutively activate isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NFκB signaling. In the present study, we show that Rac1b overexpression correlates with colorectal cancer stage with the highest levels identified in metastatic cancer. We show that Rac1b expression is associated with increased growth of colon adenocarcinoma cells and that modulation of Rac1b expression in colon adenocarcinoma cells reduces NFκB activity. The use of a Rac inhibitor in colon cancer cells reduces the 2D and 3D colony formation of colon adenocarcinoma cells, reduces the migratory capacity of these cells, and suppresses NFκB signaling. Rac inhibition also reduces tumor growth in a xenograft model of colon adenocarcinoma cells. Surprisingly, Rac1b expression and NFκB activity was upregulated in colon carcinoma cells treated with 5-FU and oxaliplatin (two components of the standard of care FOLFOX chemotherapy regimen), suggesting that Rac1b facilitates chemo-resistance by activation of NFκB signaling. Knock down of Rac1b in colon cancer cells or Rac inhibitor increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac in the colon cancer cells prevents the increase in NFκB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Moreover, while Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in significant further reduction of tumor growth than either single agent alone. Thus, we provide the first evidence that Rac1b expression confers resistance to standard of care chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NFκB signaling providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer. Citation Format: Pallavi Chaturvedi, Erik Goka, Adriana De La Garza, Dayrelis Mesa Lopez, Marc E. Lippman. Rac1b overexpression confers resistance to chemotherapy treatment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4824.