Search

Supplementary Figures 1-7. Supplementary Figure 1. (A) Capan-2 animal protocol. Supplementary Figure 2. Effects of the combination of ZA with nab-paclitaxel on the mRNA levels of alpha-SMA and Rap1A in vivo. Supplementary Figure 3. (A) Reduction of pancreatic tumor volume by Gem alone or ZA alone. Supplementary Figure 4. Cell viability assessed by MTS assay. Supplementary Figure 5. Isobologram analysis. Supplementary Figure 6. Combination of ZA with nab-paclitaxel decrease fibronectin levels in vitro and laminin levels in vivo. Supplementary Figure 7. Mechanistic association of ZA and nab-paclitaxel.

Hematologic effects of daily metronomic (MET) nab-paclitaxel.

The purpose of this study was to investigate the antitumor effects of a combination of metronomic doses of a novel delivery vehicle, PLGA-PRINT nanoparticles containing docetaxel, and antiangiogenic mEZH2 siRNA incorporated into chitosan nanoparticles. In vivo dose-finding studies and therapeutic experiments were conducted in well-established orthotopic mouse models of epithelial ovarian cancer. Antitumor effects were determined on the basis of reduction in mean tumor weight and number of metastatic tumor nodules in the animals. The tumor tissues from these in vivo studies were stained to evaluate the proliferation index (Ki67), apoptosis index (cleaved caspase 3), and microvessel density (CD31). The lowest dose of metronomic regimen (0.5 mg/kg) resulted in significant reduction in tumor growth. The combination of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA showed significant antitumor effects in the HeyA8 and SKOV3ip1 tumor models (P < 0.05). Individual as well as combination therapies showed significant antiangiogenic, antiproliferative, and proapoptotic effects, and combination therapy had additive effects. Metronomic delivery of PLGA-PRINT-docetaxel combined with CH-mEZH2 siRNA has significant antitumor activity in preclinical models of ovarian cancer. Mol Cancer Ther; 13(7); 1750–7. ©2014 AACR.

PTEN is known to be frequently mutated in uterine cancer and also dephosphorylates FAK. Here, we examined the impact of PTEN alterations on the response to treatment with a FAK inhibitor (GSK2256098). In vitro and in vivo therapeutic experiments were carried out using PTEN-mutated and PTEN-wild-type models of uterine cancer alone and in combination with chemotherapy. Treatment with GSK2256098 resulted in greater inhibition of pFAKY397 in PTEN-mutated (Ishikawa) than in PTEN-wild-type (Hec1A) cells. Ishikawa cells were more sensitive to GSK2256098 than the treated Hec1A cells. Ishikawa cells were transfected with a wild-type PTEN construct and pFAKY397 expression was unchanged after treatment with GSK2256098. Decreased cell viability and enhanced sensitivity to chemotherapy (paclitaxel and topotecan) in combination with GSK2256098 was observed in Ishikawa cells as compared with Hec1a cells. In the Ishikawa orthoptopic murine model, treatment with GSK2256098 resulted in lower tumor weights and fewer metastases than mice inoculated with Hec1A cells. Tumors treated with GSK2256098 had lower microvessel density (CD31), less cellular proliferation (Ki67), and higher apoptosis (TUNEL) rates in the Ishikawa model when compared with the Hec1a model. From a large cohort of evaluable patients, increased FAK and pFAKY397 expression levels were significantly related to poor overall survival. Moreover, PTEN levels were inversely related to pFAKY397 expression. These preclinical data demonstrate that PTEN-mutated uterine cancer responds better to FAK inhibition than does PTEN wild-type cancer. Therefore, PTEN could be a biomarker for predicting response to FAK-targeted therapy during clinical development. Mol Cancer Ther; 14(6); 1466–75. ©2015 AACR.

Mean mouse weights in orthotopic mouse models of uterine cancer.

Supplementary Figure Legends, Materials and Methods, and Table 1. Supplementary Figure Legends, Supplementary Material and Methods for RNA isolation, cDNA synthesis, and quantification of RNA species, Supplementary Table 1. Primer Sequences.

PDF - 312K, Storage stability of PLGA-PRINT nanoparticles.

There is growing recognition of the important role of metronomic chemotherapy in cancer treatment. On the basis of their unique antiangiogenic effects, we tested the efficacy of nab-paclitaxel, which stimulates thrombospondin-1, and topotecan, which inhibits hypoxia-inducible factor 1-α, at metronomic dosing for the treatment of ovarian carcinoma. In vitro and in vivo SKOV3ip1, HeyA8, and HeyA8-MDR (taxane-resistant) orthotopic models were used to examine the effects of metronomic nab-paclitaxel and metronomic topotecan. We examined cell proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (microvessel density, MVD) in tumors obtained at necropsy. In vivo therapy experiments demonstrated treatment with metronomic nab-paclitaxel alone and in combination with metronomic topotecan resulted in significant reductions in tumor weight (62% in the SKOV3ip1 model, P < 0.01 and 96% in the HeyA8 model, P < 0.03) compared with vehicle (P < 0.01). In the HeyA8-MDR model, metronomic monotherapy with either cytotoxic agent had modest effects on tumor growth, but combination therapy decreased tumor burden by 61% compared with vehicle (P < 0.03). The greatest reduction in MVD (P < 0.05) and proliferation was seen in combination metronomic therapy groups. Combination metronomic therapy resulted in prolonged overall survival in vivo compared with other groups (P < 0.001). Tube formation was significantly inhibited in RF-24 endothelial cells exposed to media conditioned with metronomic nab-paclitaxel alone and media conditioned with combination metronomic nab-paclitaxel and metronomic topotecan. The combination of metronomic nab-paclitaxel and metronomic topotecan offers a novel, highly effective therapeutic approach for ovarian carcinoma that merits further clinical development. Mol Cancer Ther; 14(12); 2677–86. ©2015 AACR.

Hematologic effects of daily metronomic (MET) nab-paclitaxel. White blood count (WBC), hemoglobin, and platelet number were assessed from tumor bearing mice after one, two, and three weeks of treatment.

PDF - 78K, Supplementary methods and legends for supplementary figures.

Mounting clinical and preclinical evidence supports a key role for sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Here we present evidence for a feed-forward loop whereby adrenergic signaling leads to increased tumoral innervation. In response to catecholamines, tumor cells produced brain-derived neurotrophic factor (BDNF) in an ADRB3/cAMP/Epac/JNK-dependent manner. Elevated BDNF levels in the tumor microenvironment increased innervation by signaling through host neurotrophic receptor tyrosine kinase 2 receptors. In patients with cancer, high tumor nerve counts were significantly associated with increased BDNF and norepinephrine levels and decreased overall survival. Collectively, these data describe a novel pathway for tumor innervation, with resultant biological and clinical implications.Significance: Sustained adrenergic signaling promotes tumor growth and metastasis through BDNF-mediated tumoral innervation. Cancer Res; 78(12); 3233–42. ©2018 AACR.

Purpose:Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity.Experimental Design: Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues.Results:Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05).Conclusions: This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target. Clin Cancer Res; 17(7); 1713–21. ©2011 AACR.

Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models.Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC).Results:ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH2-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis.Conclusion: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Supplementary Figure 1 Functional genomic analysis of HeyA8 and SKOV3ip ovarian cancer cells 4 h after exposure to norepinephrine and compared to control cells. Supplementary Figure 2 A, Western blot analysis of ADRB1, ADRB2, ADRB3, and DUSP1 expression in 10 epithelial ovarian cancer cell lines and one breast cancer cell line (MDA-231), using non-transformed ovarian epithelium cells (HIO-180) as control. B, DUSP1 mRNA levels of cells exposed to norepinephrine 10 μM for 4 h. Supplementary Figure 3 A, DUSP1 silencing. Two commercially available siRNAs that directly target the DUSP1 gene were transfected to norepinephrine-treated HeyA8 cells. B, DUSP1 overexpression. SKOV3ip1 cells were transiently transfected with a DUSP1-myc/DDK-tagged expression vector (Origene). Supplementary Figure 4 SKOV3ip1 cells were treated with paclitaxel alone (10nM) or in combination with norepinephrine (NE; 10μM) or vascular endothelial growth factor (VEGF; 10ng/ml or 50ng/ml). NE or VEGF was administered 30 minutes prior to paclitaxel exposure.

Purpose: The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457.Experimental Design: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models.Results:In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values < 0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P ≤ 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values < 0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by ∼3-fold (P < 0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis.Conclusions: Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.

Supplementary Figures S1-S5.

Supplementary Data from Therapeutic Targeting of ATP7B in Ovarian Carcinoma

CARS imaging of ex vivo ovarian cancer samples

Fig. S1. Sustained adrenergic signaling increases nerve counts in tumors. Fig. S2. Characterization of tumoral innervation. Fig. S3. NE induces BDNF expression. Fig. S4. NE-induced BDNF expression is mediated by ADRB3/Epac/Jnk. Fig. S5. BDNF increases nerve counts. Fig. S6. Adrenergic-mediated mTrkB activation leads to increased in vivo tumor nodule counts. Table S1. Alteration in pathways associated with neuronal growth and function after NE treatment (HeyA8 and SKOV3ip1 cells). Table S2. Association of Clinicopathologic variables with BDNF protein expression. Table S3. Association of Clinicopathologic variables with Nerve Counts.

Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth.Experimental Design: Expression of dopamine receptors (DR1–DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP).Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation.Conclusions: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement. Clin Cancer Res; 17(11); 3649–59. ©2011 AACR.

Purpose: VEGF-targeted therapies have modest efficacy in cancer patients, but acquired resistance is common. The mechanisms underlying such resistance are poorly understood.Experimental Design: To evaluate the potential role of immune cells in the development of resistance to VEGF blockade, we first established a preclinical model of adaptive resistance to anti-VEGF therapy. Additional in vitro and in vivo studies were carried out to characterize the role of macrophages in such resistance.Results: Using murine cancer models of adaptive resistance to anti-VEGF antibody (AVA), we found a previously unrecognized role of macrophages in such resistance. Macrophages were actively recruited to the tumor microenvironment and were responsible for the emergence of AVA resistance. Depletion of macrophages following emergence of resistance halted tumor growth and prolonged survival of tumor-bearing mice. In a macrophage-deficient mouse model, resistance to AVA failed to develop, but could be induced by injection of macrophages. Downregulation of macrophage VEGFR-1 and VEGFR-3 expression accompanied upregulation of alternative angiogenic pathways, facilitating escape from anti-VEGF therapy.Conclusions: These findings provide a new understanding of the mechanisms underlying the modest efficacy of current antiangiogenesis therapies and identify new opportunities for combination approaches for ovarian and other cancers. Clin Cancer Res; 23(22); 7034–46. ©2017 AACR.

Purpose: Chronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biologic effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer.Experimental Design: Increased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used.Results: Catecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared with control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP–PLC–PKC–CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P = 0.049) and progression-free (P = 0.0005) survival.Conclusions: These findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management. Clin Cancer Res; 22(7); 1713–24. ©2015 AACR.

S Figure 1 (A) C57BL/6 mice that received intra-peritoneal injections of IG10 cells were randomly assigned to one of two groups (control or B20 treatment). S Figure 2 (A) Bar graph represents the CD11b+/F4/80+ macrophages in tumor tissue by FACS analysis. S Figure 3 Anti-VEGF therapyââ,¬â€œresistant macrophages have heightened viability, migration, and invasion. S Figure 4 (A) Bar graph represents the percentage of viability of B20 treated macrophages. S Figure 5. VEGFR1-positive macrophages increase the effectiveness of anti-VEGF therapy.

CARS imaging of ex vivo ovarian cancer samples

Table S1

E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD- scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.

Sustained adrenergic stimulation by norepinephrine (NE) contributes to ovarian carcinoma metastasis and impairment of chemotherapy response. Although the effect of sustained NE stimulation in cancer progression is well established, less is known about its role in cancer initiation. To determine the extent to which stress hormones influence ovarian cancer initiation, we conducted a long-term (> 3 months; > 40 population doublings) experiment in which normal immortalized fallopian tube secretory (iFTSEC283) and ovarian surface epithelial (iOSE11) cell lines and their isogenic pairs containing a p53 mutation (iFTSEC283p53R175H; iOSE11p53R175H), were continuously exposed to NE (100 nM, 1 μM, 10 μM). Fallopian tube cells displayed a p53-independent increase in proliferation and colony-forming ability in response to NE, while ovarian surface epithelial cells displayed a p53-independent decrease in both assays. Fallopian tube cells with mutant p53 showed a mild loss of chromosomes and TP53 status was also a defining factor in transcriptional response of fallopian tube cells to long-term NE treatment.

Background: Anxiety and depression symptoms are known to increase cancer symptom burden, yet little is known about the longitudinal integrations of these among Hispanic/Latinx patients. The goal of this study was to explore the trajectory and longitudinal interactions among anxiety and depression, cancer symptom burden, and health-related quality of life in Hispanic/Latinx cancer patients undergoing chemotherapy. Methods: Baseline behavioral assessments were performed before starting chemotherapy. Follow-up behavioral assessments were performed at 3, 6, and 9 months after starting chemotherapy. Descriptive statistics, chi-square tests, Fisher’s exact tests, and Mann–Whitney tests explored associations among outcome variables. Adjusted multilevel mixed-effects linear regression models were also used to evaluate the association between HADS scores, follow-up visits, FACT—G scale, MDASI scale, and sociodemographic variables. Results: Increased cancer symptom burden was significantly related to changes in anxiety symptoms’ scores (adjusted β^ = 0.11 [95% CI: 0.02, 0.19]. Increased quality of life was significantly associated with decreased depression and anxiety symptoms (adjusted β^ = −0.33; 95% CI: −0.47, −0.18, and 0.38 adjusted β^= −0.38; 95% CI: −0.55, −0.20, respectively). Conclusions: Findings highlight the need to conduct periodic mental health screenings among cancer patients initiating cancer treatment.

On 7 January 2020, the southern region of Puerto Rico was struck by a 6.4 magnitude earthquake, followed by continual seismic activity. Our team performed secondary analyses to explore the relationship between exposure to seismic activity, protection (support) received, and barriers to health care access for cancer patients. Methods: The research team collected data from the database of a longitudinal case-control cohort parent study concerning the impact of Hurricane Maria in Puerto Rican cancer patients. The participants from the parent study were recruited in community clinics. The extracted data was collected from 51 cancer patients who completed the parent study’s interviews from January–July 2020 (seismic activity period). Barriers to health care were assessed using the Barrier to Care Questionaries (BCQ), which is composed of five subscales: skills, marginalization, knowledge and beliefs expectations, and pragmatics. Exposure to seismic activity and protection was assessed using their respective subscales from the Scale of Psychosocial Impact of Disasters. Results: The results showed a significant relationship between exposure to seismic activity and barriers to health care (p < 0.001) and its five subscales (p < 0.01). These results shed light on potential access to care barriers that could hinder cancer patient treatment in the event of a natural disaster.

Cancer is the leading cause of death in Puerto Rico (PR). Hurricane Maria (HM) and its aftermath lead to widespread devastation on the island, including the collapse of the healthcare system. Medically fragile populations, such as cancer survivors, were significantly affected. The goal of this study was to assess the impact of HM on barriers to care, emotional distress, and inflammatory biomarkers among cancer survivors in PR. This exploratory longitudinal study was conducted in health care facilities and community support groups from PR. Cancer survivors (n = 50) and non-cancer participants (n = 50) completed psychosocial questionnaires and provided blood samples that were used to assess inflammatory cytokines levels. Among this cohort, we identified 41 matched cancer survivors/non-cancer participants pairs. Data were analyzed through descriptive, frequencies, correlational, and regression analyses. Cancer survivors that were affected by HM reported increased barriers in accessing medical care, which were directly associated with anxiety, perceived stress, and post-traumatic symptomatology. Moreover, being a cancer survivor, predicted more barriers to receiving health care, especially in the first six weeks after the event, after which the effect was attenuated. Several inflammatory cytokines, such as CD31, BDNF, TFF3, Serpin E-1, VCAM-1, Vitamin D BP, and PDGF-AA, were significantly upregulated in cancer survivors while MMP9 and Osteopontin both had significant positive correlations with barriers to care. HM significantly impacted Puerto Ricans psychosocial well-being. Cancer survivors had significant barriers to care and showed increased serum inflammatory cytokines but did not show differences in anxiety, stress, and post-traumatic symptoms compared to non-cancer participants.

BackgroundChronic stress may affect metabolism of amino acids, lipids, and other small molecule metabolites, but these alterations may differ depending on tissue evaluated. We examined metabolomic changes in plasma and ovarian tissue samples from female mice due to chronic stress exposure.MethodsAt 12 weeks old, healthy, female, C57 black mice were randomly assigned to three weeks of chronic stress using daily restraint (2 hours/day; n=9) or normal care (n=10). Metabolomic profiling was conducted on plasma and ovarian tissues. Using the Wilcoxon Rank Test, Metabolite Set Enrichment Analysis, and Differential Network Analysis we identified metabolomic alterations occurring in response to restraint stress. All p-values were corrected for multiple testing using the false discovery rate approach.ResultsIn plasma, individual lysophosphatidylcholines (positively) and the metabolite classes carnitines (positively), diacylglycerols and triacylglycerols (inversely) were associated with restraint stress (adjusted-p’sConclusionWe identified differences in multiple lipid and amino acid metabolites in plasma and ovarian tissue of female mice after exposure to chronic stress. Some affected metabolites (primarily triacylglycerols and diacylglycerols) exhibited opposite associations with chronic stress in plasma (a marker of systemic influences) versus in ovarian tissue (representing local changes), suggesting research to understand the biological impact of chronic stress needs to consider both systemic and tissue-specific alterations.

Objectives A cancer diagnosis increases stress hormones and leads to altered psychological states. Work from our team suggests that chronic stress promotes an increased inflammatory response. Preliminary data show an altered CD4+/CD8+ T-cell ratio and a heterogeneous expression of exhaustion markers in patients with high-grade serous ovarian cancer (HGSOC). Therefore, we hypothesized that chronic stress results in loss of effector T-cell response and increased exhaustion. Methods We obtained ascites samples from 66 patients with HGSOC and measured cytokine levels using a comprehensive cytokine/chemokine magnetic bead panel. Metanephrine (an epinephrine metabolite) levels from ascites were measured by ELISA. CD8+ T-cells isolated from OC patient ascites were stimulated with epinephrine and flow cytometry was used to measure co-expression of CD38 activation marker and Granzyme B, an essential mediator of CD8+ T-cell killing capacity. Results Showed a significant increase in inflammatory cytokines in chemo-resistant and recurrent tumors: Eotaxin (p≤0.002), IL-6 (p≤0.003), and IL-7 (p≤0.009). Metanephrine, was positively correlated with pro-tumoral and inflammatory cytokines: SCD40L (p=0.032), FGF-2 (p=0.033) and MIP1a (p=0.03). Ascites-derived CD8+ T-cells treated with epinephrine, showed a decreased co-expression CD38 and Granzyme B (p=0.004). These results suggest a role for stress hormones in T-cell activity suppression. Conclusions Chemo-resistant and recurrent tumors were associated with increased pro-inflammatory cytokines. Similarly, high metanephrine levels correlated with higher pro-tumoral cytokines. Epinephrine stimulation decreased CD8+ T-cell function in ascites of HGSOC patients. These data suggest a role for stress in immunosuppression and may impact efficacy of therapies that aim to restore T-cell function.

The editors are publishing this note to alert readers to concerns about [this article][1] ([1][2]). Four sets of identical representative images exist: Figure 2A Tunel-DR2/Ctrl and Fig. 2B Tunel-DR2/Ctrl siRNA; Fig. 2A CD31/Ctrl and Fig. 2B CD31/Ctr siRNA; Fig. 2A CD31/DA and Fig. 2B CD31/DA; and

Ovarian cancer (OC) is the deadliest form of gynecological malignancies highlighted by a five-year survival rate of less than 49%. Late diagnosis and chemoresistance represent the main hurdle in the effort to improve OC patient outcomes. Currently, immunotherapy is studied as an alternative to front-line therapy, but its use in the clinic remains limited due to its modest impact on patient outcomes. It is well known that the neuroendocrine system can modulate immune cell behavior. This study aims to determine how behavioral factors, such as chronic stress and an associated increase in stress hormone levels, impact T-cell infiltration and function in ovarian cancer. As T-cells play a central role in cancer recognition and killing, we hypothesize that stress hormones could decrease the efficacy of checkpoint blockade by inducing changes in T-cell populations and contribute to T-cell exhaustion (determined by the expression of PD-1, TIM-3, FOXP3, and CTLA-4). We obtained tumor samples from C57/BL6 ID8 tumor-bearing mice subjected to daily restraint stress to dissect the interaction between the neuroendocrine system and tumor-associated T-cells. Unstressed mice were used as controls. Tumors from both groups were collected and preserved with paraffin for histological assessments. Immunohistochemistry analyses were used to evaluate the presence of CD4+ and CD8+ T-cells. To measure specific CD4+ T-cell subtype, regulatory T-cells (T-reg), FOXP3 was used as a marker. In addition, we assessed for T-cell surface markers PD-1, CTLA-4, and TIM-3. Results show a significant increase in PD1+ expressing cells (2.35x-fold; p= 0.0173) in tumors from the stress group. On the other hand, there was a non-significant decrease in CD4+ T-cells (0.78x-fold; p=0.4286) and non-significant increase in CD8+ T-cells (1.75x-fold; p= 0.1833) between groups. Although we did not observe significant differences in FOX-P3 (1.4x-fold; p=0.3152), we did observe a trend towards increased expression of this transcription factor, suggesting a predominance of T-regs in the tumor microenvironment. When comparing the presence of surface markers, there was a non-significant increase in TIM-3 (1.56x-fold; p=0.5273) and CTLA-4 (1.32x-fold; p=0.5303) positive cells in the tumor microenvironment of samples from the stress group. These data suggest that the neuroendocrine system may modulate T-cell activity, specifically the expression of PD-1 ligand. Furthermore, sustained release of stress hormones may promote an immunosuppressive tumor microenvironment, leading to T-cell exhaustion, immune evasion, and tumor progression. Citation Format: Camily Morales-López, Alexandra Aquino-Acevedo, Melanie E. Cruz-Robles, Melanie Ortiz-León, Elvin R. Hernández-Cordero, Yadiel A. Rivera-López, Margarita Rivera-Bonilla, Rebecca A. Previs, Guillermo N. Armaiz-Pena. The effect of chronic stress in T-cell population and function in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6120.

A cancer diagnosis increases stress hormones levels and leads to altered psychological states. In the context of ovarian cancer (OC), chronic stress promotes tumor growth, chemoresistance and modulates immune cell populations in the tumor microenvironment (TME). Moreover, previous work from our team suggests that chronic stress promotes an increased inflammatory response in OC. Our data show an altered CD4+/CD8+ T-cell ratio and a heterogeneous expression of exhaustion markers in patients with high-grade serous ovarian cancer (HGSOC). Therefore, we hypothesized that chronic stress results in chronic inflammation and an immunosuppressed TME. To address this, we obtained ascites from 66 patients with HGSOC and measured cytokine levels using a comprehensive Cytokine/Chemokine immunoassay. Cortisol, corticosterone, and stress hormone metabolites (metanephrine and normetanephrine) levels from ascites were measured by ELISA. CD8+ T-cells isolated from OC patient ascites were stimulated with epinephrine, and flow cytometry was used to measure co-expression of CD38 activation marker and Granzyme B, an essential mediator of CD8+ T-cell killing capacity. To further establish the impact of chronic stress on tumor progression, we subjected IG10 or ID8 tumor-bearing C57/BL6 female mice to daily restraint stress. Mice were sacrificed 8-12 weeks after inoculation, data collected, and ascites stored. Results showed an increase in inflammatory cytokines (Eotaxin, IL-6, and IL-7) in recurrent tumors of HGSOC patients. IP-10 and IFN-γ were negatively associated with cortisol levels. Moreover, normetanephrine levels positively correlated with inflammatory cytokines: IL-6, MCP-1, MCP-3, VEGF, GRO, and IL-7. Metanephrine was also positively correlated with inflammatory cytokines: SCD40L, FGF-2, and MIP1α. Ascites-derived CD8+ T-cells treated with epinephrine showed decreased co-expression CD38 and Granzyme B. Additionally, data show that daily restraint stress led to increased tumor growth in ID8 and IG10 syngeneic mouse models of OC. Ascites derived from our animal models suggest a positive correlation of inflammatory cytokines VEGF, TNFα and Eotaxin, and corticosterone levels in ID8 tumor-bearing mice. These results suggest a role for stress hormones in inflammation and immunosuppression. In conclusion, inflammatory cytokines are upregulated in recurrent HGSOC ascites samples. Cytokines that regulate T-cell function were negatively associated with cortisol levels, while stress hormone metabolites correlated with higher inflammatory cytokines. Moreover, epinephrine stimulation decreased ascites-derived CD8+ T-cell function. Inflammatory cytokines correlated with corticosterone levels in ID8 tumor-bearing mice. Overall, these data suggest a role for chronic stress in inflammation and immunosuppression, impacting the efficacy of therapies that aim to restore T-cell function. Citation Format: Alexandra N. Aquino-Acevedo, Hope Knochenhauer, Melanie Ortiz-León, Yadiel A. Rivera-López, Margarita Bonilla-Claudio, John S. Yi, Rebecca A. Previs, Guillermo N. Armaiz-Pena. Chronic stress promotes tumor-associated inflammation in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2543.

Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling-induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.

There is growing evidence that links chronic stress to ovarian cancer (OC) progression. Cancer diagnosis, chemotherapy, and other traumatic life events can lead to altered psychological states, such as chronic stress. Chronic stress induces sustained activation of the sympathetic nervous system (SNS), releasing stress hormones that modulate physiological responses across different systems, including the immune system. Specifically, chronic SNS activation alters the distribution of T-cells, thus influencing clinical outcomes. The aim of this work was to determine the effects of restraint stress on a syngeneic mouse model of OC and characterize its effects on T-cell responses. We hypothesized that restraint stress would lead to accelerated growth of existing tumors and deregulation T-cell subpopulations, promoting an immunosuppressed tumor microenvironment. To determine the effect of daily restraint stress on tumor progression, we used 8 to 12-week-old female C57/BL6 mice. After three days of daily restraint stress, mice were injected with IG10 or ID8 (1 × 10^6 cells/ 100μL) murine ovarian cancer cells. Mice were sacrificed 8–12 weeks after inoculation. Once sacrificed, tumors were removed and weighted. Our results indicate that daily restraint stress increased OC growth in both IG10/ID8 groups. Moreover, IG10 tumor-bearing mice showed increased ascites production. These results suggest an association between psychological factors and disease progression. Future experiments will assess the effects of chronic stress on T-cell maturation, effector functions, exhaustion, and activation in these samples. Results obtained from this project will support future in vivo experiments regarding the effect of restraint stress and immunotherapy efficacy.

Simple Summary Biobehavioral disorders can negatively impact patients with ovarian cancer. Growing evidence suggests that chronic stress can promote tumor progression, the release of inflammatory mediators, and macrophage infiltration into the tumor. However, the role of stress hormones in regulating cancer cell/macrophage crosstalk remains unclear. This study aimed to assess the role of stress hormone-stimulated macrophages in modulating inflammatory networks and ovarian cancer biology. Our data show that stress hormones induced secretion of inflammatory proteins in ovarian cancer cell/macrophage co-cultures. Furthermore, we show that restraint stress leads to cancer growth, macrophage infiltration, and PDGF-AA protein expression in animal models of ovarian cancer. Conversely, zoledronic acid was able to prevent the effects of restraint stress on ovarian cancer growth. Overall, our data suggest a role for stress hormone-stimulated macrophages in ovarian cancer progression and suggest the involvement of PDGF-AA as a key mediator of this process. Abstract Multiple studies suggest that chronic stress accelerates the growth of existing tumors by activating the sympathetic nervous system. Data suggest that sustained adrenergic signaling can induce tumor growth, secretion of pro-inflammatory cytokines, and macrophage infiltration. Our goal was to study the role of adrenergic-stimulated macrophages in ovarian cancer biology. Cytokine arrays were used to assess the effect of adrenergic stimulation in pro-tumoral cytokine networks. An orthotopic model of ovarian cancer was used to assess the in vivo effect of daily restraint stress on tumor growth and adrenergic-induced macrophages. Cytokine analyses showed that adrenergic stimulation modulated pro-inflammatory cytokine secretion in a SKOV3ip1 ovarian cancer cell/U937 macrophage co-culture system. Among these, platelet-derived growth factor AA (PDGF-AA), epithelial cell-derived neutrophil-activating peptide (ENA-78), Angiogenin, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-5 (IL-5), Lipocalin-2, macrophage migration inhibitory factor (MIF), and transferrin receptor (TfR) were upregulated. Enriched biological processes included cytokine-mediated signaling pathways and positive regulation of cell proliferation. In addition, daily restraint stress increased ovarian cancer growth, infiltration of CD68+ macrophages, and expression of PDGF-AA in orthotopic models of ovarian cancer (SKOV3ip1 and HeyT30), while zoledronic acid, a macrophage-depleting agent, abrogated this effect. Furthermore, in ovarian cancer patients, high PDGFA expression correlated with worse outcomes. Here, it is shown that the adrenergic regulation of macrophages and PDGFA might play a role in ovarian cancer progression.