Background/ Synopsis:Cholesterol plays an important role in sustaining tumor growth and metastasis in a large variety of cancers. New and powerful cholesterol-lowering drugs are being used in combination to treat cardiovascular diseases. Thus, it becomes intuitive to verify whether these drugs could be combined to induce a cholesterol shortage sufficient to impede tumor progression. Circulating levels of the targets of a new generation of lipid-lowering drugs have not been fully investigated in cancers. Given that drugs directed against PCSK9 (evolocumab, alirocumab, inclisiran), ANGPTL3 (evinacumab) and Lp(a) (pelacarsen) are available, it becomes important to assess circulating levels of these drug targets and their role in cancers. Objective/Purpose: To compare circulating levels of PCSK9, ANGPTL3, and Lp(a) in women with stage III breast cancer versus women with premalignant or benign breast lesions. Methods: Twenty-three plasma samples from women diagnosed with a stage III breast cancer (ductal, lobular or mixed) were matched for age with twenty-three plasma samples from women bearing premalignant (stage 0, n = 9) or benign (n = 14) breast lesions. The lipid profile (Apo B, total cholesterol, HDL cholesterol and triglycerides levels) and Lp(a) were measured on a Roche Modular analytical platform, whereas LDL levels were calculated with the Friedewald formula. ANGPTL3 and PCSK9 plasma levels were quantitated by ELISA. All statistical analyses were performed using SAS software version 9.4. Results: PCSK9 levels were significantly higher in women with stage III breast cancer compared to age-matched counterparts presenting a benign lesion (95.9 +/- 27.1 ng/mL vs. 78.5 +/- 19.3 ng/mL, p < 0.05, n = 14). Moreover, PCSK9 levels positively correlated with breast disease severity (benign, stage 0, stage III) (Rho = 0.34, p < 0.05, n = 46). In contrast, ANGPTL3 and Lp(a) plasma levels did not display any association with breast disease status and lipids did not correlate with disease severity. Conclusion: In this cohort of 46 women, PCSK9 levels increased along with the severity of breast disease. Given that PCSK9 plays an important role in maintaining cholesterolemia and has been shown to degrade MHC-I on tumor cells, impeding immune T-cells response to tumors, the association between PCSK9 levels and breast disease severity needs to be further investigated in larger studies.

Objectives This study was an in-depth exploration of unique data from a nationally representative sample of adults living in the United States to identify biomarkers associated with musculoskeletal pain. Methods We performed secondary analyses of 2003–2004 NHANES data. After a first screening of 187 markers, analyses of 31 biomarkers were conducted on participants aged ≥20 years identified in all counties using the 2000 Census Bureau data (n = 4,742). To assess the association of each biomarker with each pain outcome (acute, subacute and chronic low back, neck, and shoulder pain), analyses were carried out using multivariable logistic regression with adjustments for sex, age and body mass index. Biomarkers were considered as continuous variables and categorized at the median of their distributions. Results Pain at any site for ≥24 hours during the past month was reported by 1,214 participants. Of these, 779 mentioned that the pain had lasted for ≥3 months (“chronic pain”). α-carotene, ascorbic acid, β-carotene, mercury and total protein had a statistically significant, inverse association with ≥2 chronic pain sites. Acrylamide, alkaline phosphatase, cadmium, cotinine, glycidamide, homocysteine, retinol, triglycerides and white blood cell count were positively associated with ≥2 chronic pain sites. Few biological markers were associated with acute and subacute pain. Conclusions This study identified some biomarkers that were strongly and consistently associated with musculoskeletal pain. These results raise new hypotheses and could have tremendous implications for advancing knowledge in the field. Research on musculoskeletal pain needs to put more effort on the biological dimension of the biopsychosocial model of pain.

Background/ Synopsis: Cholesterol plays an important role in sustaining tumor growth and metastasis in a large variety of cancers. New and powerful cholesterol-lowering drugs are being used in combination to treat cardiovascular diseases. Thus, it becomes intuitive to verify whether these drugs could be combined to induce a cholesterol shortage sufficient to impede tumor progression. Circulating levels of the targets of a new generation of lipid-lowering drugs have not been fully investigated in cancers. Given that drugs directed against PCSK9 (evolocumab, alirocumab, inclisiran), ANGPTL3 (evinacumab) and Lp(a) (pelacarsen) are available, it becomes important to assess circulating levels of these drug targets and their role in cancers. Objective/Purpose: To compare circulating levels of PCSK9, ANGPTL3, and Lp(a) in women with stage III breast cancer versus women with premalignant or benign breast lesions. Methods: Twenty-three plasma samples from women diagnosed with a stage III breast cancer (ductal, lobular or mixed) were matched for age with twenty-three plasma samples from women bearing premalignant (stage 0, n=9) or benign (n= 14) breast lesions. The lipid profile (Apo B, total cholesterol, HDL cholesterol and triglycerides levels) and Lp(a) were measured on a Roche Modular analytical platform, whereas LDL levels were calculated with the Friedewald formula. ANGPTL3 and PCSK9 plasma levels were quantitated by ELISA. All statistical analyses were performed using SAS software version 9.4. Results: PCSK9 levels were significantly higher in women with stage III breast cancer compared to age-matched counterparts presenting a benign lesion (95.9 +/- 27.1 ng/mL vs. 78.5 +/- 19.3 ng/mL, p<0.05, n=14). Moreover, PCSK9 levels positively correlated with breast disease severity (benign, stage 0, stage III) (Rho=0.34, p<0.05, n=46). In contrast, ANGPTL3 and Lp(a) plasma levels did not display any association with breast disease status and lipids did not correlate with disease severity. Conclusion: In this cohort of 46 women, PCSK9 levels increased along with the severity of breast disease. Given that PCSK9 plays an important role in maintaining cholesterolemia and has been shown to degrade MHC-I on tumor cells, impeding immune T-cells response to tumors, the association between PCSK9 levels and breast disease severity needs to be further investigated in larger studies.

Background/ Synopsis: Cholesterol plays an important role in sustaining tumor growth and metastasis in a large variety of cancers. New and powerful cholesterol-lowering drugs are being used in combination to treat cardiovascular diseases. Thus, it becomes intuitive to verify whether these drugs could be combined to induce a cholesterol shortage sufficient to impede tumor progression. Circulating levels of the targets of a new generation of lipid-lowering drugs have not been fully investigated in cancers. Given that drugs directed against PCSK9 (evolocumab, alirocumab, inclisiran), ANGPTL3 (evinacumab) and Lp(a) (pelacarsen) are available, it becomes important to assess circulating levels of these drug targets and their role in cancers. Objective/Purpose: To compare circulating levels of PCSK9, ANGPTL3, and Lp(a) in women with stage III breast cancer versus women with premalignant or benign breast lesions. Methods: Twenty-three plasma samples from women diagnosed with a stage III breast cancer (ductal, lobular or mixed) were matched for age with twenty-three plasma samples from women bearing premalignant (stage 0, n=9) or benign (n= 14) breast lesions. The lipid profile (Apo B, total cholesterol, HDL cholesterol and triglycerides levels) and Lp(a) were measured on a Roche Modular analytical platform, whereas LDL levels were calculated with the Friedewald formula. ANGPTL3 and PCSK9 plasma levels were quantitated by ELISA. All statistical analyses were performed using SAS software version 9.4. Results: PCSK9 levels were significantly higher in women with stage III breast cancer compared to age-matched counterparts presenting a benign lesion (95.9 +/- 27.1 ng/mL vs. 78.5 +/- 19.3 ng/mL, p<0.05, n=14). Moreover, PCSK9 levels positively correlated with breast disease severity (benign, stage 0, stage III) (Rho=0.34, p<0.05, n=46). In contrast, ANGPTL3 and Lp(a) plasma levels did not display any association with breast disease status and lipids did not correlate with disease severity. Conclusion: In this cohort of 46 women, PCSK9 levels increased along with the severity of breast disease. Given that PCSK9 plays an important role in maintaining cholesterolemia and has been shown to degrade MHC-I on tumor cells, impeding immune T-cells response to tumors, the association between PCSK9 levels and breast disease severity needs to be further investigated in larger studies.

e17583 Background: Cancer cells require important amounts of cholesterol to sustain their growth. High-grade serous ovarian carcinomas (HGSOC), as an aggressive form of ovarian cancer, are no exception. Mechanisms allowing HGSOC to secure their lipid supplies are not yet fully elucidated. Recent years have highlighted ANGPTL3, PCSK9 and Apo CIII as key players in lipid metabolism. To date, impact of HGSOC on these key lipid-regulating factors is scantly documented. Herein, we compared ANGPTL3, PCSK9, Apo CIII and Lp(a) levels in women with HGSOC versus benign ovarian lesion (BOL) to better understand lipid metabolism in HGSOC. Methods: Plasma samples from 31 women with a HGSOC and 40 women with a BOL were assayed for ANGPTL3, PCSK9, and Apo CIII levels by ELISA. The lipid panel, Apo B and Lp(a) levels were measured on a Roche Modular analytical platform. Results: Higher levels of ANGPTL3 was observed in HGSOC (84 ng/ml, SD ±29 ng/ml, n = 31) vs. BOL (67 ng/ml, SD ±31 ng/ml, n = 40); HGSOC vs BOL p = 0.019. Receiver operating characteristic (ROC) curve analyses for ANGPTL3 combined with age indicated an area under curve of 67.7% (p = 0.005). Partial correlations indicated associations between the lipid panel and ANGPTL3, but only for BOL. Conclusions: In this cohort of 71 women, ANGPTL3 levels were higher in patients with HGSOC. This finding requires further validation in a larger cohort of HGSOC patients.

e12547 Background: Cholesterol plays an important role in sustaining tumor growth and metastasis in a large variety of cancers. New and powerful cholesterol-lowering drugs are being used in combination to treat cardiovascular diseases. As drugs directed against PCSK9 (evolocumab, alirocumab, inclisiran), ANGPTL3 (evinacumab) and Lp(a) (pelacarsen) are available, it becomes of interest to assess the circulating levels of these drug targets and their role in cancers. Our purpose here is to compare circulating levels of PCSK9, ANGPTL3, and Lp(a) in women with stage III breast cancer versus women with a premalignant or benign breast lesion. Methods: Twenty-three plasma samples from women diagnosed with a stage III breast cancer (ductal, lobular or mixed) were matched for age with twenty-three plasma samples from women bearing premalignant (stage 0, n=9) or benign (n= 14) breast lesions. Lipid profile (Apo B, total cholesterol, HDL and triglyceride levels) and Lp(a) were measured on a Roche Modular analytical platform. LDL levels were calculated with the Friedewald formula. ANGPTL3 and PCSK9 plasma levels were quantitated by ELISA. Difference between breast disease groups was deemed significant when group-comparison generated p-values < 0.05. Results: PCSK9 levels were significantly higher in women with stage III breast cancer compared to age-matched counterparts presenting a benign lesion (95.9 +/- 27.1 ng/mL vs. 78.5 +/- 19.3 ng/mL, p<0.05, n=14). Moreover, PCKS9 levels positively correlated with breast disease severity (benign, stage 0, stage III) (Rho=0.34, p<0.05, n=46). In contrast, ANGPTL3, Lp(a) or lipid plasma levels did not display any correlation with breast disease severity. Conclusions: In this cohort of 46 women, PCSK9 levels increased along with the severity of breast disease. Given that PCSK9 plays an important role in maintaining cholesterolemia and has been shown to degrade MHC-I on tumor cells, impeding immune T-cells response to tumors, confirmation of PCSK9 elevation in a larger cohort would pave the way for trials studying lipid-lowering drugs as adjuvant treatments in breast cancer.

Mammary epithelial cells (MECs) are known to have their metabolism reprogrammed following pregnancy to allow the increased energy requirements for lactation. The estrogen receptor α (ERα) is mainly associated with the regulation of biological pathways linked to mammary gland development but its influence on MECs metabolism is still unknown. Our hypothesis is that ERα reprograms cell metabolism in normal MECs, a phenomenon that would be reprogrammed during breast carcinogenesis. Few in vitro models are used to study MECs, and most of them do not express ERα. Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs to study ERα metabolic functions using mass spectrometry (MS). Purified MECs from nulliparous mice remain differentiated for up to six days with &gt;85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favouring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. To further understand this reprogramming, stable isotope tracer analysis in primary culture organoids are currently performed. In addition, pregnancy and breast-feeding are known to be protective against breast carcinogenesis. Consequently, we also performed MEC purification and organoid culture using mammary glands from multiparous mice. Intriguingly, organoid phenotypic characterization indicated a difference in organoid structures between MECs from nulliparous and multiparous mice. Furthermore, we observe significant differences in estrogenic response between both conditions, suggesting that pregnancy and/or lactation promotes the establishment of specific epigenetic marks that are preserved even ex vivo. Chromatin immunoprecipitation of specific histone marks and MS-based metabolic studies are ongoing to better understand the different responses of mammary organoids to estrogens between nulliparous and multiparous mice. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures and for MS-based metabolomics. We demonstrated that these organoids retain a functional ERα pathway over time and that ERα significantly reprograms multiple metabolic pathways. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis. Citation Format: Aurélie Lacouture, Cynthia Jobin, Alisson Clemenceau, Cindy Weidmann, Line Berthiaume, Dominic Bastien, Isabelle Laverdière, Martin Pelletier, Caroline Diorio, Francine Durocher, Étienne Audet-Walsh. A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-01.

Breast cancer is the leading cause of cancer-related death in women worldwide. Our group previously performed a human transcriptome array on normal tissue, atypical ductal hyperplasia, ductal carcinoma in situ and estrogen receptor-α positive invasive ductal carcinoma. Among the transcripts down-regulated during carcinogenesis: Secreted Frizzled-Related Protein 1 (SFRP1), a Wnt signaling pathway antagonist inversely associated with age-related lobular involution has been identified.Interestingly, SFRP1 expression is higher in presence of hydroxyapatite microcalcifications in non-tumoral breast tissue. SFRP1 is also known to be involved in the increase of bone resorption, notably by downregulating osteoblastic differentiation. This suggests that the lack of SFRP1 could be involved in both osteoblast-like phenotype acquisition and early breast carcinogenesis. Unfortunately, data on the mechanism of microcalcifications formation and their impacts on breast carcinogenesis are still lacking.We hypothesize that loss of SFRP1 is responsible for microcalcifications accumulation and early breast carcinogenesis. Our objectives are:1. To define the impact of microcalcifications on SFRP1 expression in non-tumoral mammary gland and 2. to decipher the causal role of SFRP1 in microcalcifications formation. Organoid culture using freshly purified epithelial cells from nulliparous and multiparous mice mammary glands, in presence or in absence of hydroxyapatite crystals has been performed. Organoids number and phenotype have been studied (aim 1). We also have modulated SFRP1 expression in mammary cell lines by lentiviral transduction and performed proliferation, migration and alizarin assays to assess the tumoral aggressivity and cells abilities to produce calcium deposits in vitro (aim 2). SFRP1 and markers of osteoblastic differentiation (Runt-related transcription factor 2, RUNX2) and bone formation (Alkaline Phosphatase, ALPL; Osteopontin, SPP1; Periostin, POSTN) will also be quantified by quantitative PCR in both mice organoids and human mammary cell lines (aims 1 and 2). 1. The ratio between the mammary gland and body weight obtained from nulliparous mice (n = 12) is significantly lower compared with multiparous mice (n = 12; fold-change = 1.84; p-value &lt; 0.001). Hydroxyapatite crystals induced an increase in the proportion of organoids with lumen and branching compared with the control without hydroxyapatite specifically in organoids obtained from nulliparous mice. Organoid’s size and gene expression profiles are still to be assessed. 2. SFRP1 downregulation in a non-tumoral atypical ductal hyperplasia cell line (MCF10AT1) induced an increase in proliferative and migratory cells abilities. It was also responsible for an increase in estrogen receptor 1 (ESR1) expression, suggesting that SFRP1 is related to luminal carcinogenesis. Results also demonstrate that decreasing SFRP1 in MCF10AT1 induced a profile similar to growth stage in bone tissue, which notably means an increase of expression of the osteoblastic transcription factor RUNX2 and the marker of mineralization SPP1, also involved in lactation-related pink adipose tissue formation. We also observed an increase of POSTN expression, known to be involved in decreased bone mineral density during lactation.SFRP1 upregulation in the invasive luminal A cell line MCF7 reduced cell migration and ESR1 expression. Likewise, it increased MCF7 proliferation compared to control. In addition, it induced an increase of RUNX2 and SPP1 expression, similar to what is reported in matrix mineralization profile in bone. These results support our hypothesis that SFRP1 has a causal role in the acquisition of a malignant phenotype but also in osteoblast-like phenotype in mammary epithelial cells. The cause of SFRP1 downregulation in women remains to be explored. Citation Format: Alisson Clemenceau, Aurélie Lacouture, Juliette Bherer, Audet-Walsh Étienne, Caroline Diorio, Francine Durocher. Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-11.