Breast cancer outcomes vary across populations, yet Native American women remain scarcely represented in tumor-genomic resources, limiting population-specific molecular insights. We generated matched somatic mutation, copy-number, and RNA-seq profiles for 17 breast tumors from Native American women and performed race-stratified comparisons with White cases from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort (TCGA-BRCA). We observed population-associated differences across molecular layers, including higher mutation frequencies in ARID1B, NOTCH4, and MHC class II genes (HLA-DRB1/HLA-DRB5) in Native American tumors, and broader CNV alterations in White tumors. Integrative analyses highlighted antigen processing/presentation and cell-adhesion pathways, with class II alterations in Native American tumors and class I gains (e.g., HLA-A/HLA-B) plus CD274 amplification in White tumors, suggesting differences in immune visibility and checkpoint modulation. We also noted contrasts in nucleotide-excision-repair involvement (ERCC5/POLE mutations vs ERCC1/CUL4A CNV gains), and mutational-signature analysis indicated greater MMR- and AID/POLE-associated exposures in the White cohort. To our knowledge, this study provides an initial multi-omics characterization of breast tumors from Native American women and offers a resource and hypotheses for larger, harmonized studies to assess prognostic and therapeutic relevance.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) associated with infection by the human papillomavirus (HPV) continues to increase and 71% of OPSCC cases in the United States are HPV+. We have previously performed microRNA profiling of HPV+ vs HPV- OPSCC and identified miR-106b as significantly elevated in HPV+ cases (Miller et al., 2015; Am J Path 185:679-92 [1]). MicroRNA target prediction identified the gene PDCD1LG2 as a highly ranked target of miR-106b. This gene corresponds to the protein programmed cell death ligand 2 (PD-L2), a high affinity ligand for the programmed cell death protein 1 (PD-1) receptor on T-cells. The objective of this study was to evaluate PD-L2 expression in a panel of HPV+ and HPV- OPSCC tumors. Using tissues from two distinct patient cohorts, results show significantly lower PD-L2 protein expression levels in HPV+ tumors. These data suggest that future research on regulation of PD-L2 expression in HPV+ OPSCC is warranted.

The mechanisms enabling disseminating tumor cells to survive and colonize peritoneal niches during ovarian cancer metastasis remain incompletely understood. Here, we present MetTag, a single-cell barcoding and transcriptome profiling approach with time-stamped batch identifiers to resolve temporal dynamics of dissemination. Sequencing of MetTag barcodes reveals enrichment of early-disseminated clones across metastatic sites, and targeted depletion of pioneers diminishes the outgrowth of later arriving cells. MetTag-coupled single-cell RNA sequencing uncovers a distinct interferon-gamma-centric transcriptional trajectory enriched among pioneer clones. CRISPR/Cas9 screening and subsequent interferon-gamma receptor 1 knockout in pioneer cells significantly reduces metastatic burden, highlighting a critical window where active interferon-gamma signaling shapes the post-seeding metastatic niche and outgrowth. Mechanistically, the tumor-intrinsic interferon-gamma response and peritoneal macrophages cooperatively shield disseminating tumor cells from anoikis. Our study defines the temporal clonal architecture of peritoneal metastasis, revealing a first come, first served dissemination principle, where pioneer fitness determines the success of subsequent colonizers. The mechanisms enabling disseminating tumour cells (DTCs) to survive and colonise peritoneal niches in ovarian cancer metastasis remain poorly understood. Using single-cell barcoding, transcriptome profiling, and in vivo CRISPR screening, the authors identify a niche-adaptive IFNγ response signature that supports pioneer DTC outgrowth.

ABSTRACT Background Cancer predominantly affects older individuals, with age being a significant risk factor for cancer incidence and metastasis. Biological sex also plays a crucial role in influencing metastasis and survival outcomes. In colorectal cancer (CRC), both the incidence and mortality from metastatic disease are higher in males relative to females. Aim The aim of this study was to use a syngeneic murine intraperitoneal (i.p.) metastasis model of CRC (MC‐38 cells) to compare disease burden between young and aged female and male mice. Methods MC‐38 cells tagged with red fluorescent protein were injected i.p. and tumor burden quantified longitudinally and at endpoint. As the peritoneal mesothelial cell is the initial site of tumor:host interaction in i.p. metastasis, primary murine peritoneal mesothelial cells were subjected to bottom up proteomic analysis to identify proteins differentially expressed among the cohorts. Results Recapitulating human epidemiological data, aged male mice exhibited the highest i.p. metastatic burden. Proteomic results identified multiple differentially expressed proteins. Protein tyrosine phosphatase 4A1 (PTP4A1), highly overexpressed in the male aged cohort relative to male young, female aged or female young, was chosen for further study. Functional analyses indicated that PTP4A1 promotes cancer:mesothelial adhesion and a small molecule inhibitor of PTP4A1, designated CMPD‐43, reduced RhoA activity and inhibited heterotypic cell adhesion. Conclusion These results provide a resource for comparative proteomics of the peritoneal mesothelial cell in sex‐ and age‐based cohorts. Functional data support further consideration of PTP4A1 as a potential therapeutic target for impeding CRC metastasis particularly in an aged male cohort.

Aging is one of the most significant risk factors for breast cancer. With the growing interest in the alterations of the aging breast tissue microenvironment, it is identified that aging is related to tumorigenesis, invasion, and drug resistance. However, current pre-clinical disease models often neglect the impact of aging and sometimes result in worse clinical outcomes. In this study, aged animal-generated materials are utilized to create and validate a novel age-mimetic breast cancer model that generates an aging microenvironment for cells and alters cells toward a more invasive phenotype found in the aged environment. Furthermore, the age-mimetic models are utilized for 3D breast cancer invasion assessment and high-throughput screening of over 700 drugs in the FDA-approved drug library. 36 potential effective drug targets as well as 34 potential drug targets with different drug responses in different age groups are identified, demonstrating the potential of this age-mimetic breast cancer model for further in-depth breast cancer studies and drug development.

Metastasis is an emergent continuum, driven by evolving reciprocal adaptations between continuously disseminating tumor cells (DTCs) and the specialized metastatic niches of distant organs. The interplay between intrinsic and niche-driven mechanisms that enables DTCs to survive and home to distant organs remains incompletely understood. Here, using MetTag, a single-cell barcoding and transcriptome profiling approach with time-stamped batch identifiers (BC.IDs) and functional CRISPR screening, we resolved the clonality, temporal dynamics, and molecular determinates of DTC colonization success across evolving metastatic niches. Deep sequencing of barcodes revealed preferred enrichment of early-disseminated clones across metastatic niches. Single-cell RNA sequencing (scRNA-seq) coupled with RNA velocity analyses in ascites and metastasis-bearing omenta uncovered an emergent and distinct interferon-gamma (IFNγ) centric transcriptional trajectory, enriched among early seeding clones. In vivo CRISPR/Cas9 screening of metastatic niche-specific signatures demonstrated that genes belonging to the IFNγ response are functionally important for peritoneal metastasis. Knockout of IFNγ receptor 1 (Ifngr1) in the first batch of DTCs significantly reduced metastatic burden and extended survival, underscoring the importance of tumor cell intrinsic IFNγ signaling in shaping post-seeding metastatic niche (PSMN) and subsequent metastatic co-evolution. Mechanistically, tumor intrinsic IFNγ response and ascites-derived tumor-associated macrophages (TAMs) protect cancer cells from anoikis-mediated death by promoting pro-survival signaling. Our study defines temporal dynamics of disseminating tumor cells at metastatic niches and reveals a general "first come, first served" pro-metastatic adaptation principle of DTCs.

Epithelial ovarian cancer (OvCa) remains a leading cause of mortality among gynecological cancers. Metastasis to the peritoneum, characterized by tumor cell adhesion to and invasion of the mesothelial lining of the abdominal cavity, represents a critical early event in OvCa metastatic progression. The median age of diagnosis is 63 and there exists a strong correlation between advanced age, OvCa incidence and disease stage. Moreover, the aged peritoneal cavity represents a permissive niche for metastatic dissemination. To investigate age-related factors that influence host-tumor communication in metastatic progression, the current study isolated small extracellular vesicles (sEVs) from the peritoneal lavage of healthy tumor-naïve young (3–6 month) and aged (20–22 month) mice. sEVs were analyzed using LC-MS/MS to identify sEV protein cargoes and incubated with murine and human OvCa cells to evaluate effect on pro-metastatic behaviors. Treatment of human or murine OvCa cells with sEVs from healthy aged hosts significantly enhanced adhesion to peritoneal mesothelial cells in a three-dimensional in vitro meso-mimetic culture assay and to the intact omentum in a short-term in vivo adhesion assay relative to OvCa cells treated with sEVs from young hosts. OvCa cell invasion of collagen gels was also enhanced by aged host-derived sEVs. Proteomic analysis of sEV protein cargos identified differentially expressed proteins in sEVs obtained from aged vs. young hosts that may play a significant role in regulation of adhesion. This was confirmed using meso-mimetic adhesion assays with function blocking antibodies or small molecule inhibitors, supporting a potential role for several proteins in promoting intra-peritoneal dissemination in the aged host. Results suggest that sEVs derived from the aged peritoneal microenvironment can contribute significantly to disease progression, highlighting sEV-mediated host: tumor communication as a potential therapeutic target for intervention in OvCa progression or recurrence in the aged host.

Abstract Obesity is a recognized non-infectious pandemic that increases ovarian cancer (OvCa) incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity (DIO) mouse model (Liu et al. Can.Res. 2015; 75:5046). DIO also elevated expression and nuclear localization of sterol regulatory element binding protein 1 (SREBP1), a master regulator of de novo lipogenesis and lipid homeostasis. A follow-up study using DIO models showed a significantly diminished response to standard-of-care (SOC) paclitaxel/carboplatin chemotherapy in high fat diet (HFD) mice relative to low fat diet (LFD) controls (Liu et al., J.Exp.Clin.Can.Res. 2023; 42:165). Both studies showed an effect of DIO on tumor fibrosis and immune landscape and these changes were also observed in the ovarian cancer tumors of women with high body mass index. In the current study, we have evaluated the effect of inhibitors of SREBP1 processing, in combination with SOC chemotherapy, in the context of DIO in pre-clinical models of OvCa. Our results show significantly diminished post-treatment tumor recurrence in HFD mice receiving SOC chemotherapy plus an SREBP1 processing inhibitor, with concomitant alterations in the peritoneal immune landscape. Together these data suggest that the reported negative impact of obesity on OvCa patient survival may be due obesity-induced changes in lipid regulatory factors (SREBP1) and the tumor microenvironment (immune landscape, fibrosis) that alter chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting SREBP1, immune cells and/or fibrosis in the obese host. Citation Format: M. Sharon Stack, Yueying Liu, Jing Yang, Tyvette S. Hilliard, Zhikun Wang, Jeff Johnson, Wanrui Wang, Gena Dominique, Elizabeth I. Harper, Nicholas M. Stavrou, Anna Juncker-Jensen. Targeting sterol regulatory element binding protein 1 (SREBP1) improves response to standard-of-care chemotherapy and alters the tumor microenvironment in pre-clinical models of obesity and ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A082.

Ovarian cancer is usually detected in the advanced stages. Existing treatments for high grade serous ovarian cancer (HGSOC) are not adequate and approximately fifty percent of patients succumb to this disease and die within five years after diagnosis. We conducted pre-clinical studies in a mouse model of ovarian cancer to evaluate disease outcome in response to treatment with the multi-kinase inhibitor cabozantinib. Cabozantinib is a receptor tyrosine kinase inhibitor with multiple targets including vascular endothelial growth factor receptor-2 (VEGFR-2), associated with immune suppression in ovarian cancer. Mice (C57BL/6) were injected with ID8-RFP ovarian tumor cells and treated with cabozantinib. Studies investigated ascites development, tumor burden and regulation of anti-tumor immunity with treatment. Mice treated with cabozantinib had significantly decreased solid tumor burden and decreased malignant ascites as compared to untreated controls. Improved outcome in cabozantinib treated mice was associated with a significantly higher percentage of CD69 early activated T cells, a higher percentage of granzyme B secreting CD8 T cells, the enhanced release of cytokines and chemokines known to recruit CD8 T cells and amplify T cell function, as well as reduced VEGFR-2. Findings suggest that cabozantinib is an important clinical agent capable of improving ovarian cancer in mice potentially in part by priming the autologous immune system to promote anti-tumor immunity.

ABSTRACT The gut microbiome changes with age and affects regions beyond the gut, including the ovarian cancer tumor microenvironment. In this review summarizing the literature on the gut microbiome in ovarian cancer and in aging, we note trends in the microbiota composition common to both phenomena and trends that are distinctly opposite. Both ovarian cancer and aging are characterized by an increase in proinflammatory bacterial species, particularly those belonging to phylum Proteobacteria and genus Escherichia, and a decrease in short‐chain fatty acid producers, particularly those in Clostridium cluster XIVa (family Lachnospiraceae) and the Actinobacteria genus Bifidobacterium. However, although beneficial bacteria from family Porphyromonadaceae and genus Akkermansia tend to increase with normal, healthy aging, these bacteria tend to decrease in ovarian cancer, similar to what is observed in obesity or unhealthy aging. We also note a lack in the current literature of research demonstrating causal relationships between the gut microbiome and ovarian cancer outcomes and research on the gut microbiome in ovarian cancer in the context of aging, both of which could lead to improvements to ovarian cancer diagnosis and treatment.

Metastatic ovarian cancer (MOC) is highly deadly, due in part to the limited efficacy of standard-of-care chemotherapies to metastatic tumors and non-adherent cancer cells. Here, we demonstrated the effectiveness of a combination therapy of GRP78-targeted (TNPGRP78pep) and non-targeted (NP) nanoparticles to deliver a novel DM1-prodrug to MOC in a syngeneic mouse model. Cell surface-GRP78 is overexpressed in MOC, making GRP78 an optimal target for selective delivery of nanoparticles to MOC. The NP + TNPGRP78pep combination treatment reduced tumor burden by 15-fold, compared to untreated control. Increased T cell and macrophage levels in treated groups also suggested antitumor immune system involvement. The NP and TNPGRP78pep components functioned synergistically through two proposed mechanisms of action. The TNPGRP78pep targeted non-adherent cancer cells in the peritoneal cavity, preventing the formation of new solid tumors, while the NP passively targeted existing solid tumor sites, providing a sustained release of the drug to the tumor microenvironment.

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.

<p>Overview of experimental cohorts.</p>

<p>Representative histology of Cohort 2 metastatic implants in control or DIO C57/Bl6 mice.</p>

Supplementary Figure Legends 1-3 from Ovarian Cancer Cell Detachment and Multicellular Aggregate Formation Are Regulated by Membrane Type 1 Matrix Metalloproteinase: A Potential Role in I.p. Metastatic Dissemination

Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Altered Gene Expression, and Oral Tumor Progression

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Legends for Supplemental Figures S1-S4

Supplementary Data including methods, legends, supplemental figures 1-5 and supplemental table 1-2. Supplemental Fig. 1: WNT5A mRNA and Wnt5a protein expression in OvCa cell lines, Supplemental Fig. 2: Silencing of WNT5A in LP9 human peritoneal mesothelial cells, Supplemental Fig. 3: WNT5A expression in murine tissues and longitudinal imaging of tumor burden in WNT5A-modified cohorts, Supplemental Fig. 4: Fgr inhibition abrogates Wnt5a pro-metastatic cellular phenotypes, Supplemental Fig. 5. Fgr and phospho-Fgr protein levels in human serous ovarian carcinoma. Supplemental Table. 1: Wnt5a shRNA lentivirus particles sequences, Supplemental Table. 2: Primers for qPCR