Primary Human Cancer Research Articles

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

Abstract A014: Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers

Abstract Endometriosis-associated carcinomas (EACs) and endometrial malignancies have many commonalities yet their pathophysiology, particularly those with high-risk histology such as clear cell, is understudied. EACs are histologically and clinically distinct from other gynecological cancers due to their unique origin within endometriosis. The endometriotic microenvironment is uniquely hypoxic with high levels of iron due to recurrent menstrual cycling, and this iron-rich microenvironment has been linked to promoting carcinogenesis through iron-induced oxidative stress. However, the mechanism by which the endometriotic microenvironment contributes to EAC tumorigenesis and how this compares with endometrial cancer is unknown. Most of our current knowledge of EACs is built on either epidemiologic analyses or correlative studies. Additionally, most studies of the endometriotic microenvironment primarily focus on the epithelial component while the role of the stroma has been largely unexplored. Mesenchymal stem cells (MSC) are a critical component of the stromal microenvironment. Recently, we identified a key role of endometriosis derived MSCs (enMSCs) in altering ovarian clear cell carcinoma iron regulation. Interestingly, enMSCs resemble the primary endometrium derived MSCs, which suggested their role in endometrial cancer. This stromal subset is characterized by the loss of surface CD10 expression. It is known that cancer cells, particularly clear cell carcinoma (CCC), have a unique requirement for high levels of iron. We hypothesize that CD10 negative enMSCs promote growth of EAC and endometrial cancer through iron regulation. MSCs were isolated from primary human benign endometriosis, benign endometrium, or endometrial cancer. Flow cytometry was used to measure surface CD10 expression. We investigated the role of CD10 negative enMSCs on CCC iron regulation. Our results showed that CD10 negative enMSCs promote CCC growth by exporting iron through the upregulation of Hephaestin (HEPH) and Ferroportin (FPO) which then led to increase labile iron pool (LIP) in CCC cells. However, increasing the LIP does not occur with iron supplementation alone, this indicates the critical role of CD10 negative enMSCs in regulating iron handling in CCC cells. Additionally, our data showed that CD10 negative enMSCs alter the balance of Ferritin L and Ferritin H- the main iron storage proteins- in CCC cells. Mechanistically, our preliminary data showed that CD10 negative enMSCs directly donate Ferritin L into CCC cells. Further, CD10 negative enMSCs enhance Ferritin H degradation through the upregulation of ferritinophagy in CCC cells. In conclusion, our results indicate there is a sub-population of enMSCs, marked by the loss of CD10 expression, that enhances EAC and endometrial cancer cell growth through iron regulation. This highlights the existence of a tumor-promoting stromal cell within both the endometrium and endometriosis which can be capitalized on as an “Achilles' heel” to uncover novel treatment targets for EAC and endometrial cancer. Citation Format: Huda I. Atiya, Lan G. Coffman. Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A014.

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.

Abstract IA023: Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer

Abstract Phenotypic plasticity is thought to assist immune evasion, metastasis, and therapeutic resistance in tumor evolution. Epigenetic mechanisms such as DNA methylation provide for a more easily acquired and flexible, yet semi-stable adaptation by the tumor cell than hardwired genetic changes. Cancer-associated gain of DNA methylation at promoter CpG islands or distal regulatory elements is associated with transcriptional silencing, whereas loss of DNA methylation is observed at late-replicating, lamina-attached regions of the genome, tracking with cumulative cell divisions. One of the most well-documented genomic features predisposing to DNA hypermethylation is Polycomb Repressive Complex (PRC) occupancy in precursor cells, consistent with an epigenetic block to cellular differentiation as an early or predisposing event in carcinogenesis. A central question is whether this epigenetic switch from reversible PRC repression to more stable DNA hypermethylation occurs before onset of clonal expansion, or whether it represents an ongoing process during tumor evolution. Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. We have developed a tumor processing pipeline to deeply and comprehensively map DNA methylation patterns in primary human colorectal cancer (CRC) and adjacent tissues, while preserving spatial information, enabling inference of the temporal order of molecular alterations. We employ a suite of technologies to map the spatial heterogeneity of DNA methylation patterns in primary human CRC. These include a deep single-cell whole-genome bisulfite sequencing technique that improves genomic coverage several-fold over existing published protocols, providing DNA methylation measurements at the majority of CpGs in the genome in individual tumor cells. We have also calibrated and validated an epigenetic clock that allows us to accurately measure the relative replicative history of individual cells. We infer phylogenetic relationships among subclones, and relate these to their spatial distribution in the primary tumor. We find that some tumors have high proportions of PRC-associated clonal hypermethylation, indicating that these hypermethylation events were already present prior to clonal expansion, possibly contributing to susceptibility to malignant transformation in the cell-of-origin, whereas other tumors appear to have active PRC-associated CpG island hypermethylation ongoing during tumor expansion, generating subclonal heterogeneity, which may influence post-therapeutic recurrence potential. Citation Format: Peter W. Laird, Nathan J. Spix, Hsiao-yun Milliron, Manpreet Kalkat, Emily Eugster, David Sokol, Emily Jung, Paula Nolte, Kelly K. Krzyzanowski, Toshinori Hinoue, Hui Shen. Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA023.

The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling.

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin-high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.

Abstract A016: LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma

Abstract Immunotherapy has emerged as a breakthrough in the improvement of survival outcomes for lung adenocarcinoma (LUAD), however effective response requires the combination of blocking inhibitory signals on the tumor surface and antigen presentation to the tumor surface for proper immune recognition. Several commercially available and robust methods exist for identification of tumors displaying immune-inhibitory surface receptors, such as PD-L1, however it is currently difficult to predict effectiveness of antigen presentation on the cell surface. To address this, we utilized clinical and next-generation sequencing data from The Cancer Genome Atlas (TCGA) to identify gene signatures that are correlated to tumor mutational burden (TMB) within cancers of epithelial origins as a surrogate for neoantigen signatures. We identified LINC00261 as a top gene correlated to TMB, whose expression activates DNA damage response pathways in vitro along with resistance to cisplatin. LINC00261 expression was also significantly correlated to MHC class I and II genes involved in endogenous neoantigen presentation expression within the TCGA-LUAD cohort. This relationship was confirmed in vitro through ectopic reintroduction of LINC00261 for key MHC class II presentation genes. Interferon gamma-induced MHC gene activation in vitro was also able to induce endogenous expression of LINC00261. The staining of primary human lung cancer sections suggested that loss of LINC00261 is associated with an immunosuppressive tumor microenvironment. Taken together, our results suggest there is a mechanistic relationship in the silencing of LINC00261 in LUAD and compromised DNA repair, accumulation of mutations, and reduced antitumor immune response. Citation Format: Jonathan Castillo, Tianchun Xue, Mayela Norwood, Samantha Joseph, Alan L. Epstein, W. Dean Wallace, Anthony W. Kim, Crystal N. Marconett. LINC00261 is functionally linked to tumor DNA mutational burden and tumor immune microenvironment composition in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A016.

NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Primary Human Papillomavirus Test Uptake and Cervical Cancer Screening Trends in the Midwest, United States.

Despite U.S. Preventive Services Task Force and American Cancer Society endorsement of primary HPV screening, limited published data shows low uptake. Assess cervical cancer screening rates over time, particularly primary HPV test uptake, among patients in a midwestern practice. Evaluate associations between sociodemographics and screening adherence. Cross-sectional study. Qualifying subjects and type of screening test used were identified by applying ICD-9, ICD-10, lab test, and CPT codes to the Unified Data Platform. Sociodemographics were found through the electronic health record. Primary HPV uptake represented <1% of annual screening from 1/2017 to 1/2022. On 1/1/2022, only 55% of 21 to 29 year old and 63% of 30 to 65 year old were up to date with screening among the studied population. For 21 to 29 year old, compared with White women, Black women were 28% less likely to be screened [RR = 0.72 (0.66-0.79)]. Compared with never-smokers, current smokers were 9% less likely to be screened [RR = 0.91 (0.87-0.96)], past smokers were 14% more likely [RR = 1.14 (1.09-1.2)]. Among 30 to 65 year old, compared with White women, Black women were 14% less likely to be screened [RR = 0.86 (0.81-0.9)]. Compared with never-smokers, current smokers were 21% less likely to be screened [RR = 0.79 (0.77-0.81)], past smokers were 6% less likely [RR = 0.94 (0.92-0.95)]. Jointly considering race, ethnicity, smoking status, Charlson score, and rurality, findings were similar for 21 to 29 year old; Black women were screened less than White women [RR = 0.73 (0.67-0.79)]; current smokers [RR = 0.9 (0.85-0.94)] and past smokers [RR = 1.12 (1.06-1.17)] were screened less than never smokers. For 30 to 65 year old, Black women were screened less than White women [RR = 0.83 (0.79-0.88)]; current smokers [RR = 0.8 (0.78-0.81)] and past smokers [RR = 0.95 (0.93-0.96)] were screened less than never smokers. Screening rates remained below the Healthy People 2030 goal of 79.2% over time, particularly for younger Black women and current smokers, with minimal use of primary HPV screening.

Metadichol, a Natural Ligand for the Expression of Yamanaka Reprogramming Factors in Human Cardiac, Fibroblast, and Cancer Cell Lines

The reprogramming of somatic cells into embryonic-like stem cells requires the activation of four essential transcription factors, referred to as the Yamanaka factors: Sox2, Oct4, Klf4, and c-Myc. However, the utilization of these genes and viral vectors for their delivery presents a risk of carcinogenesis, which renders induced pluripotent stem cell (iPSC) lines unsuitable for clinical applications. Typically, direct reprogramming involves the use of viral vectors to induce the expression of these factors. In contrast, metadichol, a novel approach, enhances the expression of Yamanaka factors in cells using a dose ranging from 1 pg to 100 ng, thereby preventing the need for viral vectors. This non-viral method renders cellular reprogramming safer and more clinically viable. Treatment with metadichol within the specific dose range resulted in a substantial augmentation of the fibroblast expression of OCT4, SOX2, KLF4, KLF2, and Nanog, which was confirmed through qRT-PCR and western blot analyses. The expression levels of OCT4, KLF4, Nanog, and Sox2, exhibited an increase of 4.01-, 3.51-, 1.26-, and 2.5-fold, respectively, compared to the controls. Notably, A549 and Colo-205 cancer cells demonstrated a marked elevation in expression levels. The reprogramming of primary human cancer cells presents considerable challenges. However, in triple-negative primary breast cancer cells, metadichol treatment led to the substantial upregulation of the expression levels of OCT4, KLF4, Nanog, and Sox2 by 19.6-, 8.07-, 2.45-, and 6.91-fold, respectively, across the dose range of 1 pg to 100 ng. Klotho, an antiaging gene that is modulated by metadichol, down regulates TP53, a crucial factor for the production of somatic cell iPSCs. Additionally, metadichol enhances the availability of vitamin C, which is essential for generating iPSCs from somatic cells. Increased levels of alkaline phosphatase, indicative of cellular differentiation, were observed in the treated group compared to the controls. Metadichol is a nontoxic nanoemulsion that is derived from long-chain C26–C28 alcohols from food sources and activates Yamanaka factors across various cell types without requiring the use of viral or CRISPR-based methodologies, thus substantially advancing somatic cell reprogramming.

Pair bonding and disruption impact lung transcriptome in monogamous Peromyscus californicus

Social interactions affect physiological and pathological processes, yet their direct impact in peripheral tissues remains elusive. Recently we showed that disruption of pair bonds in monogamous Peromyscus californicus promotes lung tumorigenesis, pointing to a direct effect of bonding status in the periphery (Naderi et al., 2021). Here we show that lung transcriptomes of tumor-free Peromyscus are altered in a manner that depends on pair bonding and superseding the impact of genetic relevance between siblings. Pathways affected involve response to hypoxia and heart development. These effects are consistent with the profile of the serum proteome of bonded and bond-disrupted Peromyscus and were extended to lung cancer cells cultured in vitro, with sera from animals that differ in bonding experiences. In this setting, the species’ origin of serum (deer mouse vs FBS) is the most potent discriminator of RNA expression profiles, followed by bonding status. By analyzing the transcriptomes of lung cancer cells exposed to deer mouse sera, an expression signature was developed that discriminates cells according to the history of social interactions and possesses prognostic significance when applied to primary human lung cancers. The results suggest that present and past social experiences modulate the expression profile of peripheral tissues such as the lungs, in a manner that impacts physiological processes and may affect disease outcomes. Furthermore, they show that besides the direct effects of the hormones that regulate bonding behavior, physiological changes influencing oxygen metabolism may contribute to the adverse effects of bond disruption.

The GLP-1 receptor is expressed in vivo by human metastatic prostate cancer.

The glucagon-like peptide-1 (GLP-1) receptor agonist, liraglutide, reduces human prostate cancer incidence, and similar GLP-1 receptor agonists reduce in vitro proliferation and in vivo growth of prostate cancer cell lines. Primary human prostate cancer expresses the GLP-1 receptor (GLP-1R) in vitro. Cancer evolves with stage, and whether advanced-stage human prostate cancer expresses GLP-1R is unknown. We hypothesised and aimed to prove that human metastatic castrate-resistant prostate cancer (mCRPC) expresses the GLP-1R in vivo. We hypothesised that mCRPC would thus be detectable by positron emission tomography/computed tomography (PET/CT) using a radiotracer bound to a GLP-1R ligand, as in exendin PET/CT. Men with mCRPC, with more than one prostate-specific membrane antigen (PSMA)-avid lesion on PET/CT scanning (pathognomic in that setting for prostate cancer lesions), were approached to undergo PET/CT with gallium68-Dota-exendin-4. We documented PET/CT PSMA-avid lesions, which were also PET/CT exendin avid, as evidence of in vivo GLP-1R expression. Out of the 24 men referred, three did not meet the inclusion criteria. Seventeen declined, largely because the study offered them no therapeutic benefit. Among the four men imaged, three had no exendin-avid lesions, while one had six osseous PSMA-avid lesions, three of which were also exendin avid. We demonstrated in vivo GLP-1R expression by human mCPRC, detecting PET/CT lesions avid for both PSMA and exendin, in one of four participants. GLP-1R expression may thus occur even in advanced-stage prostate cancer. Our data contribute to growing evidence supporting the testing of GLP-1 receptor agonists for therapeutic benefit in prostate cancer.

Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers

BackgroundIn addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials.MethodsWe prospectively performed multiparameter flow cytometry and single-cell RNA sequencing (scRNA-Seq) paired with TCR sequencing on freshly resected human primary tumors of various histological types to precisely determine ICP expression levels within T-cell subsets.ResultsWithin a given tumor type, we found high inter-individual variability for tumor infiltrating CD45 + cells and for T-cells subsets. The proportions of CD8+ T-cells (~ 40%), CD4+ FoxP3- T-cells (~ 40%) and CD4+ FoxP3+ T-cells (~ 10%) were consistent across patients and indications. Intriguingly, both stimulatory (CD25, CD28, 4-1BB, ICOS, OX40) and inhibitory (PD-1, CTLA-4, PD-L1, CD39 and TIGIT) checkpoint proteins were predominantly co-expressed by intratumoral CD4+FoxP3+ T-cells. ScRNA-Seq paired with TCR sequencing revealed that T-cells with high clonality and high ICP expressions comprised over 80% of FoxP3+ cells among CD4+ T-cells. Unsupervised clustering of flow cytometry and scRNAseq data identified subsets of CD8+ T-cells and of CD4+ FoxP3- T-cells expressing certain checkpoints, though these expressions were generally lower than in CD4+ FoxP3+ T-cell subsets, both in terms of proportions among total T-cells and ICP expression levels.ConclusionsTumor histology alone does not reveal the complete picture of the tumor immune contexture. In clinical trials, assumptions regarding target expression should rely on more sensitive and specific techniques than conventional IHC or transcriptomics. Flow cytometry and scRNAseq accurately characterize ICP expression within immune cell subsets. Much like in hematology, flow cytometry can better describe the immune contexture of solid tumors, offering the opportunity to guide patient treatment according to drug target expression rather than tumor histological type.

Anti-inflammatory mechanisms in cancer research: Characterization of a distinct M2-like macrophage model derived from the THP-1 cell line.

Macrophages play an essential role in cancer development. Tumor-associated macrophages (TAMs) have predominantly M2-like attributes that are associated with tumor progression and poor patient survival. Numerous methods have been reported for differentiating and polarizing macrophages invitro, but there is no standardized and validated model for creating TAMs. Primary cells show varying cytokine responses depending on their origin and functional studies utilizing these cells may lack generalization and validity. A distinct cell line-derived TAM-like M2 subtype is required to investigate the mechanisms mediated by anti-inflammatory TAMs invitro. Our previous work demonstrated a standardized protocol for creating an M2 subtype derived from a human THP-1 cell line. The cell expression profile, however, has not been validated. The aim of this study was to characterize and validate the TAM-like M2 subtype macrophage created based on our protocol to introduce them as a standardized model for cancer research. Using qRT-PCR and ELISA, we demonstrated that proinflammatory, anti-inflammatory, and tumor-associated marker expression changed during THP-1-derived marcrophage development invitro, mimicking a TAM-related profile (e.g., TNFα, IL-1β). The anti-inflammatory marker IL-8/CXCL8, however, is most highly expressed in young M0 macrophages. Flow cytometry showed increased expression of CD206 in the final TAM-like M2 macrophage. Single-cell RNA-sequencing analysis of primary human monocytes and colon cancer tissue macrophages demonstrated that cell line-derived M2 macrophages resembled a TAM-related gene profile. The THP-1-derived M2 macrophage based on a standardized cell line model represents a distinct anti-inflammatory TAM-like phenotype with an M2a subtype profile. This model may provide a basis for invitro investigation of functional mechanisms in a variety of anti-inflammatory settings, particularly colon cancer development.

Assessment of Primary Human Liver Cancer Cells by Artificial Intelligence-Assisted Raman Spectroscopy.

We investigated the possibility of using Raman spectroscopy assisted by artificial intelligence methods to identify liver cancer cells and distinguish them from their Non-Tumor counterpart. To this aim, primary liver cells (40 Tumor and 40 Non-Tumor cells) obtained from resected hepatocellular carcinoma (HCC) tumor tissue and the adjacent non-tumor area (negative control) were analyzed by Raman micro-spectroscopy. Preliminarily, the cells were analyzed morphologically and spectrally. Then, three machine learning approaches, including multivariate models and neural networks, were simultaneously investigated and successfully used to analyze the cells' Raman data. The results clearly demonstrate the effectiveness of artificial intelligence (AI)-assisted Raman spectroscopy for Tumor cell classification and prediction with an accuracy of nearly 90% of correct predictions on a single spectrum.

P16/Ki67 dual stain triage versus cytology in primary human papillomavirus-based cervical cancer screening with limited genotyping.

The introduction of primary human papillomavirus (HPV) cervical cancer screening requires the implementation of an appropriate triage strategy that will be effective in detecting high-grade cervical disease without losing diagnostic specificity. From the 30.066 screening tests results, a total of 1086 with available high-risk human papillomavirus (HRHPV) with limited genotyping, cytology, and p16/Ki67 dual-stain were selected. Two triage strategies for primary HPV screening were analyzed retrospectively based on the study group. Performance characteristics for p16/Ki67 and cytology triage in the detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) were calculated, detected in colposcopic biopsy. In HPV16/18-positive cases, primary HPV with p16/Ki67 triage was significantly more specific than cytology (53.1%/16.8% for CIN2+; p < 0.0001; 45.9%/17.0% for CIN3+; p < 0.0001), with yielded sensitivity (95.7%/84.8% for CIN2+; p = 0.0955; 100.0%/87.5% for CIN3+; p = 0.0832). In other HRHPV-positive cases (N16/N18), p16/Ki67 triage was also significantly higher specific (51.3%/15.3% for CIN2+; p < 0.0001; 44.5%/16.5% for CIN3+; p < 0.0001), with sensitivity (92.3%/74.4% for CIN2+; p = 0.0522; 90.9%/81.8% for CIN3+; p = 0.5637). Diagnostic predictive values were significantly higher for p16/Ki67 triage with the highest PPV in HPV16/18-positive cases for CIN2+ (45.4%; 95% confidence interval [CI]: 35.2-55.8; p < 0.0001) and very high NPV in all HPV-positive cases regardless of detected genotype (96.3%-100.0%). The risk (1-NPV) for CIN3+ in HRHPV16/18-positive/p16/Ki67-negative women was 0.0%. Superior diagnostic performance compared to cytology for detecting cervical cancer precursors indicates that p16/Ki67 dual-immunostain may be a highly effective tool of triage in primary HPV screening with limited HPV 16/18 genotyping in secondary cervical cancer prevention.

Muscarinic receptor agonist-induced βPix binding to β-catenin promotes colon neoplasia

M3 muscarinic receptors (M3R) modulate β-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.

Reducing the Invasiveness of Low- and High-Grade Endometrial Cancers in Both Primary Human Cancer Biopsies and Cell Lines by the Inhibition of Aquaporin-1 Channels.

Aquaporin (AQP) channels in endometrial cancer (EC) cells are of interest as pharmacological targets to reduce tumor progression. A panel of compounds, including AQP1 ion channel inhibitors (AqB011 and 5-(phenoxymethyl) furan-2-carbaldehyde, PMFC), were used to test the hypothesis that inhibition of key AQPs can limit the invasiveness of low- and high-grade EC cells. We evaluated the effects on transwell migration in EC cell lines (Ishikawa, MFE-280) and primary EC cells established from surgical tissues (n = 8). Quantitative PCR uncovered classes of AQPs not previously reported in EC that are differentially regulated by hormonal signaling. With estradiol, Ishikawa showed increased AQPs 5, 11, 12, and decreased AQPs 0 and 4; MFE-280 showed increased AQPs 0, 1, 3, 4, 8, and decreased AQP11. Protein expression was confirmed by Western blot and immunocytochemistry. AQPs 1, 4, and 11 were colocalized with plasma membrane marker; AQP8 was intracellular in Ishikawa and not detectable in MFE-280. AQP1 ion channel inhibitors (AqB011; PMFC) reduced invasiveness of EC cell lines in transwell chamber and spheroid dispersal assays. In Ishikawa cells, transwell invasiveness was reduced ~41% by 80 µM AqB011 and ~55% by 0.5 mM 5-PMFC. In MFE-280, 5-PMFC inhibited invasion by ~77%. In contrast, proposed inhibitors of AQP water pores (acetazolamide, ginsenoside, KeenMind, TGN-020, IMD-0354) were not effective. Treatments of cultured primary EC cells with AqB011 or PMFC significantly reduced the invasiveness of both low- and high-grade primary EC cells in transwell chambers. We confirmed the tumors expressed moderate to high levels of AQP1 detected by immunohistochemistry, whereas expression levels of AQP4, AQP8, and AQP11 were substantially lower. The anti-invasive potency of AqB011 treatment for EC tumor tissues showed a positive linear correlation with AQP1 expression levels. In summary, AQP1 ion channels are important for motility in both low- and high-grade EC subtypes. Inhibition of AQP1 is a promising strategy to inhibit EC invasiveness and improve patient outcomes.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

Adipocytes reprogram cancer cell metabolism by diverting glucose towards glycerol-3-phosphate thereby promoting metastasis.

In the tumor microenvironment, adipocytes function as an alternate fuel source for cancer cells. However, whether adipocytes influence macromolecular biosynthesis in cancer cells is unknown. Here we systematically characterized the bidirectional interaction between primary human adipocytes and ovarian cancer (OvCa) cells using multi-platform metabolomics, imaging mass spectrometry, isotope tracing and gene expression analysis. We report that, in OvCa cells co-cultured with adipocytes and in metastatic tumors, a part of the glucose from glycolysis is utilized for the biosynthesis of glycerol-3-phosphate (G3P). Normoxic HIF1α protein regulates the altered flow of glucose-derived carbons in cancer cells, resulting in increased glycerophospholipids and triacylglycerol synthesis. The knockdown of HIF1α or G3P acyltransferase 3 (a regulatory enzyme of glycerophospholipid synthesis) reduced metastasis in xenograft models of OvCa. In summary, we show that, in an adipose-rich tumor microenvironment, cancer cells generate G3P as a precursor for critical membrane and signaling components, thereby promoting metastasis. Targeting biosynthetic processes specific to adipose-rich tumor microenvironments might be an effective strategy against metastasis.

Isorhapontigenin (ISO) inhibits malignant cell transformation, migration, and invasion through MEG3/NEDD9 signaling in Cr(VI)-transformed cells

Cr(VI) compounds are confirmed human carcinogens. Maternally expression 3 (MEG3) is the first long non-coding RNA to be identified as a tumor suppressor. MEG3 is frequently downregulated or lost in various primary human tumor tissues and cancer cell lines. Downregulation of MEG3 is associated with cancer initiation, progression, and metastasis. Our previous study has revealed that MEG3 was lost and NEDD9 was upregulated in Cr(VI)-transformed cells compared to those in passage-matched normal BEAS-2B cells. Overexpression of MEG3 reduced NEDD9. β-Catenin was activated in Cr(VI)-transformed cells, overexpression of MEG3 or knockdown of NEDD9 inhibited the activation of β-Catenin. The results from the present study showed that isorhapontigenin (ISO) treatment is able to suppress cell proliferation, migration, and invasion of Cr(VI)-transformed cells. Further study showed that ISO treatment in Cr(VI)-transformed cells decreases the levels of Ki67, a biomarker for cell proliferation, and of cyclin D1, a regulator for the cell cycle. ISO elevated the MEG3 expression level in Cr(VI)-transformed cells. The DNA methylation transferases DNMT3a, DNMT3b, and DNMT1 levels were reduced upon ISO treatment. ISO treatment decreased both mRNA and protein levels of NEDD9. In addition, ISO treatment reduced the activation of β-catenin. Slug was upregulated and E-Cadherin was downregulated in Cr(VI)-transformed cells, treatment with ISO decreased Slug and increased E-Cadherin. This study demonstrated that ISO is a potent therapeutical agent against lung cancer induced by Cr(VI).