A new Neu-a syngeneic model of spontaneously metastatic HER2-positive breast cancer.

Metastatic disease results from the dissemination of tumor cells beyond their organ of origin to grow in distant organs and is the primary cause of death in patients with advanced breast cancer. Preclinical murine models in which primary tumors spontaneously metastasize are valuable tools for studying metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. The NT2.5-lung metastasis (-LM) cell line was derived from serial passaging of tumor cells that were macro-dissected from spontaneous lung metastases after orthotopic mammary implantation of parental NT2.5 cells. Within one week of NT2.5-LM implantation, metastases are observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. We demonstrate that NT2.5-LM metastases are positive for NeuN-the murine equivalent of human epidermal growth factor 2 (HER2). We further demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT), suggestive of their enhanced metastatic potential. Genomic analyses support these findings and reveal enrichment in EMT-regulating pathways. In addition, the metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. The new NT2.5-LM model provides certain advantages over the parental NT2/NT2.5 model, given its more rapid and spontaneous development of metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses.

A comprehensive single-cell breast tumor atlas defines epithelial and immune heterogeneity and interactions predicting anti-PD-1 therapy response.

We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use T cell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. T cell InteractPrint was predictive of response in both trials versus PD-L1 (AUC = 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

Mimicking the breast metastatic microenvironment: characterization of a novel syngeneic model of HER2+ breast cancer.

Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.

Antibody-drug conjugates in breast cancer: overcoming resistance and boosting immune response.

Antibody-drug conjugates (ADCs) have emerged as a revolutionary therapeutic class, combining the precise targeting ability of monoclonal antibodies with the potent cytotoxic effects of chemotherapeutics. Notably, ADCs have rapidly advanced in the field of breast cancer treatment. This innovative approach holds promise for strengthening the immune system through antibody-mediated cellular toxicity, tumor-specific immunity, and adaptive immune responses. However, the development of upfront and acquired resistance poses substantial challenges in maximizing the effectiveness of these therapeutics, necessitating a deeper understanding of the underlying mechanisms. These mechanisms of resistance include antigen loss, derangements in ADC internalization and recycling, drug clearance, and alterations in signaling pathways and the payload target. To overcome resistance, ongoing research and development efforts are focused on urgently identifying biomarkers, integrating immune therapy approaches, and designing novel cytotoxic payloads. This Review provides an overview of the mechanisms and clinical effectiveness of ADCs, and explores their unique immune-boosting function, while also highlighting the complex resistance mechanisms and safety challenges that must be addressed. A continued focus on how ADCs impact the tumor microenvironment will help to identify new payloads that can improve patient outcomes.

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

Improving the odds together: a framework for breast cancer research scientists to include patient advocates in their research.

Including patient advocates in basic cancer research ensures that breast cancer research is intentional, supports effective communication with broader audiences, and directly connects researchers with those who they are striving to help. Despite this utility, many cancer research scientists do not work with patient advocates. To understand barriers to engagement and build a framework for enhanced interactions in the future, we hosted a workshop with patient advocates and researchers who do engage, then discussed findings at an international metastatic breast cancer conference to solicit additional feedback and suggestions. Findings demonstrate that researchers are uncertain about how to initiate and maintain relationships with advocates. We offer actionable steps to support researchers working with patient advocates to improve cancer research and accomplish our collective goal of improving lives of those who have been diagnosed with breast cancer. We hope that this initiative will facilitate such collaborative efforts.

Organoid Co-culture Methods to Capture Cancer Cell-Natural Killer Cell Interactions.

Metastasis is a complex process that has been historically difficult to model in culture. Host immune responses play critical roles in restraining and promoting metastatic tumor cells. Here we describe a method of 3D organotypic co-culture of natural killer cells and tumor organoids to capture interactions between the two cellular populations. These assays can be used to model key aspects of metastatic biology and to screen for the effectiveness of agents that stimulate natural killer cell cytotoxicity.

The changing role of natural killer cells in cancer metastasis.

Natural killer (NK) cells are innate immune cells that are critical to the body's antitumor and antimetastatic defense. As such, novel therapies are being developed to utilize NK cells as part of a next generation of immunotherapies to treat patients with metastatic disease. Therefore, it is essential for us to examine how metastatic cancer cells and NK cells interact with each other throughout the metastatic cascade. In this Review, we highlight the recent body of work that has begun to answer these questions. We explore how the unique biology of cancer cells at each stage of metastasis alters fundamental NK cell biology, including how cancer cells can evade immunosurveillance and co-opt NK cells into cells that promote metastasis. We also discuss the translational potential of this knowledge.

Olaparib Use in Patients With Metastatic Breast Cancer Harboring Somatic BRCA1/2 Mutations or Mutations in Non-BRCA1/2, DNA Damage Repair Genes.

BACKGROUND: Poly-ADP ribose polymerase (PARP) inhibitors (PARPi) are active in patients with germline BRCA1/2 (gBRCA1/2)-mutated breast cancer, accounting for 5% to 10% of all breast cancers. Another 5% to 10% harbor somatic BRCA1/2 (sBRCA1/2) mutations or mutations in non-BRCA1/2, homologous recombination repair (HRR) genes but until recently, there were no data for the use of PARPi in these patients. This study examines the use of olaparib in patients with metastatic breast cancer harboring sBRCA1/2 or germline or somatic non-BRCA1/2, HRR mutations and demonstrates potential activity of PARPi in this setting. METHODS: In this retrospective, single institution study, patients who were treated with off-label, off-protocol olaparib for metastatic breast cancer harboring sBRCA1/2 or germline or somatic non-BRCA1/2, HRR mutations were identified. The primary aim was to describe these patients' demographics, tumor characteristics, mutations, safety and tolerability, response rates, progression free survival, PARPi-associated survival and subsequent treatment. RESULTS: Seven patients were treated off-label, off-trial with olaparib for sBRCA1/2-mutated cancers (n = 4) or non-BRCA1/2, HRR-mutated cancers (n = 3). All patients with sBRCA1/2-mutated cancers responded to PARP inhibition; patients with non-BRCA1/2, HRR-mutated cancers did not respond. The median progression free survival in patients with a sBRCA1/2 mutation was 6.5 months (range 5-9 months) vs. 3 months (range 2-4 months) in patients with non-BRCA1/2, HRR mutations. CONCLUSION: This single institution experience adds to recent larger reports confirming evidence for PARPi therapy in patients with metastatic breast cancer harboring sBRCA1/2 mutations. No activity was observed in patients with either germline or somatic non-BRCA1/2, HRR-mutated cancers.

Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.

Rare Germline Genetic Variants and the Risks of Epithelial Ovarian Cancer.

A family history of ovarian or breast cancer is the strongest risk factor for epithelial ovarian cancer (EOC). Germline deleterious variants in the BRCA1 and BRCA2 genes confer EOC risks by age 80, of 44% and 17% respectively. The mismatch repair genes, particularly MSH2 and MSH6, are also EOC susceptibility genes. Several other DNA repair genes, BRIP1, RAD51C, RAD51D, and PALB2, have been identified as moderate risk EOC genes. EOC has five main histotypes; high-grade serous (HGS), low-grade serous (LGS), clear cell (CCC), endometrioid (END), and mucinous (MUC). This review examines the current understanding of the contribution of rare genetic variants to EOC, focussing on providing frequency data for each histotype. We provide an overview of frequency and risk for pathogenic variants in the known susceptibility genes as well as other proposed genes. We also describe the progress to-date to understand the role of missense variants and the different breast and ovarian cancer risks for each gene. Identification of susceptibility genes have clinical impact by reducing disease-associated mortality through improving risk prediction, with the possibility of prevention strategies, and developing new targeted treatments and these clinical implications are also discussed.

Cancer cells educate natural killer cells to a metastasis-promoting cell state.

Natural killer (NK) cells have potent antitumor and antimetastatic activity. It is incompletely understood how cancer cells escape NK cell surveillance. Using ex vivo and in vivo models of metastasis, we establish that keratin-14+ breast cancer cells are vulnerable to NK cells. We then discovered that exposure to cancer cells causes NK cells to lose their cytotoxic ability and promote metastatic outgrowth. Gene expression comparisons revealed that healthy NK cells have an active NK cell molecular phenotype, whereas tumor-exposed (teNK) cells resemble resting NK cells. Receptor-ligand analysis between teNK cells and tumor cells revealed multiple potential targets. We next showed that treatment with antibodies targeting TIGIT, antibodies targeting KLRG1, or small-molecule inhibitors of DNA methyltransferases (DMNT) each reduced colony formation. Combinations of DNMT inhibitors with anti-TIGIT or anti-KLRG1 antibodies further reduced metastatic potential. We propose that NK-directed therapies targeting these pathways would be effective in the adjuvant setting to prevent metastatic recurrence.

Impact of the Current US Preventive Services Task Force Recommendations for Cervical Cancer Screening in Young Women 21 to 29 Years Old.

OBJECTIVES: In 2012, the US Preventive Services Task Force decreased the recommended frequency of cervical cytology screening to once every 3 years and recommended against testing women younger than 21 years regardless of sexual history. We evaluated the impact of this in 21 to 29-year-old women at a tertiary care academic medical center in 2011 and 2017. METHODS: We retrospectively analyzed Papanicolaou test results at two time points in 21- to 29-year-old women. RESULTS: There was a decrease in the number of high-grade lesions in 21- to 25-year-old women (odds ratio [OR], 0.36) from 2011 to 2017. Within the 26- to 29-year-old patient group, there was a trend toward a higher percentage of high-grade squamous intraepithelial lesion (HSIL) in 2017 compared to 2011 on cytology, which did not reach statistical significance (OR, 1.46). However, follow-up histologic specimens showed a higher percentage of HSIL in 2017 compared to 2011 in this age group (OR, 2.16). CONCLUSIONS: Our findings suggest that the cervical cancer screening guidelines introduced in 2012 have not had a detrimental impact on the outcomes of cervical cancer screening for 21- to 25-year-old women. However, we need to continue monitoring the effects of decreased screening in 26- to 29-year-old women.

Immunotherapy for Merkel cell carcinoma: a turning point in patient care.

Merkel Cell carcinoma (MCC) is a rare but aggressive cancer, with an estimated disease-associated mortality as high as 46%. MCC has proven to be an immunologically responsive disease and the advent of immune checkpoint inhibitors has changed the treatment landscape for patients with advanced MCC. In this review, we discuss the rationale for the use of immune checkpoint inhibition, review current single agent therapies tested in and approved for MCC, and discuss emerging immunotherapeutic options for these patients.

Itch Management: Physical Approaches (UV Phototherapy, Acupuncture).

BACKGROUND: Physical therapies refer to non-medical treatment strategies, including surgery, cryotherapy, UV phototherapy, and acupuncture. Most physical approaches are inappropriate in the context of itch. UV phototherapy and acupuncture may be effective in the management of itch. METHODS: A literature search was performed using MEDLINE and EMBASE. Bibliographies were reviewed for relevant articles. RESULTS: Narrowband UVB (311-313 nm) and UVA1 (340-400 nm) are equally effective in managing atopic dermatitis and associated itch. The efficacy of broadband UVB in reducing uraemic itch has been demonstrated in a series of randomised controlled trials, but more recent studies have failed to reproduce these results. Non-randomised, uncontrolled studies and case series suggest that UV is effective in managing itch associated with cholestasis, chronic urticaria, prurigo, cutaneous T-cell lymphoma, aquagenic itch, and scleroderma. UV phototherapy is well tolerated, and no significant relationship between UVB therapy and skin cancer has been found. Experimentally, acupuncture has been shown to reduce allergen-related itch, although this finding has been limited by the small number of studies, inconsistency in agreement on acupuncture sites and study design, small sample sizes, and limited follow-up. CONCLUSIONS: UV phototherapy is an effective treatment for itch associated with atopic dermatitis. UVB may be effective in managing itch associated with end-stage kidney disease, cholestasis, chronic urticaria, prurigo, cutaneous T-cell lymphoma, aquagenic itch, and scleroderma. Phototherapy should be combined with standard first-line therapies. Insufficient evidence exists to justify acupuncture as a physical therapy for itch. Further well-designed studies are required to establish the effectiveness of physical therapies in managing itch.

Alcohol activates the hedgehog pathway and induces related procarcinogenic processes in the alcohol-preferring rat model of hepatocarcinogenesis.

BACKGROUND: Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls. METHODS: Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC. RESULTS: Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues. CONCLUSIONS: Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis.

Macrophage-derived Hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni.

BACKGROUND: Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. AIMS: Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. METHODS: Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. RESULTS: Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. CONCLUSION: SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.