Tumors Developed Research Articles

When Do Tumours Develop? Neoplastic Processes Across Different Timescales: Age, Season and Round the Circadian Clock.

While it is recognised that most, if not all, multicellular organisms harbour neoplastic processes within their bodies, the timing of when these undesirable cell proliferations are most likely to occur and progress throughout the organism's lifetime remains only partially documented. Due to the different mechanisms implicated in tumourigenesis, it is highly unlikely that this probability remains constant at all times and stages of life. In this article, we summarise what is known about this variation, considering the roles of age, season and circadian rhythm. While most studies requiring that level of detail be done on humans, we also review available evidence in other animal species. For each of these timescales, we identify mechanisms or biological functions shaping the variation. When possible, we show that evolutionary processes likely played a role, either directly to regulate the cancer risk or indirectly through trade-offs. We find that neoplastic risk varies with age in a more complex way than predicted by early epidemiological models: rather than resulting from mutations alone, tumour development is dictated by tissue- and age-specific processes. Similarly, the seasonal cycle can be associated with risk variation in some species with life-history events such as sexual competition or mating being timed according to the season. Lastly, we show that the circadian cycle influences tumourigenesis in physiological, pathological and therapeutic contexts. We also highlight two biological functions at the core of these variations across our three timescales: immunity and metabolism. Finally, we show that our understanding of the entanglement between tumourigenic processes and biological cycles is constrained by the limited number of species for which we have extensive data. Improving our knowledge of the periods of vulnerability to the onset and/or progression of (malignant) tumours is a key issue that deserves further investigation, as it is key to successful cancer prevention strategies.

Small-molecule targeting BCAT1-mediated BCAA metabolism inhibits the activation of SHOC2-RAS-ERK to induce apoptosis of Triple-negative breast cancer cells

IntroductionTriple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with the worst prognosis. Exploring novel carcinogenic factors and therapeutic drugs for TNBC remains a focus to improve prognosis. Branched-chain amino acid transaminase 1 (BCAT1), a crucial enzyme in branched-chain amino acid (BCAA) metabolism, has been linked to various tumor developments, but its carcinogenic function and mechanism in TNBC remain unclear. Eupalinolide B (EB) is a naturally-derived small-molecule with anti-tumor activity, but its role in TNBC remains unknown. ObjectivesBy exploring the targets and pharmacological mechanisms of EB in inhibiting TNBC, this study aimed to discover novel therapeutic targets and potential inhibitors for TNBC, and elucidate novel pathogenic mechanisms of TNBC. MethodsThe inhibitory effect of EB on TNBC was investigated using mouse models and cellular phenotypic experiments. Activity-based protein profiling (ABPP) technology, pull down-WB, CETSA-WB and MST were utilized to discover and validate the targets of EB. The oncogenic role of BCAT1 was determined through clinical data analysis and biochemical experiments. To elucidate the mechanism by which EB inhibited TNBC, many methods, including but not limited to HPLC and proteomic sequencing were used. ResultsWe found that EB significantly inhibited TNBC progression. We identified BCAT1 as the direct target of EB and confirmed that BCAT1 was critical for TNBC development. EB inhibited BCAT1-involved BCAA metabolism to reduce the synthesis of BCAAs (including Leu, Ile, and Val), thereby inhibiting SHOC2 (a Leu-rich repeat protein) expression and the downstream SHOC2-participating RAS-ERK signaling pathway, ultimately leading to apoptosis of TNBC cells. ConclusionCollectively, this study not only elucidates the oncogenic role of BCAT1 and its downstream SHOC2-RAS-ERK signaling axis in TNBC progression but also opens up avenues for potential therapies targeting BCAT1 or BCAA metabolism (using EB alone or in combination with its inhibitor candesartan) for TNBC treatment.

Abstract B044: Durable T-cell-mediated anti-tumor immune response to pancreatic cancer cells overexpressing interleukin 6

Abstract Tumor immune resistance contributes to low survivorship in patients with pancreatic ductal adenocarcinoma (PDAC). To understand how tumor-secreted factors might impact anti-tumor immunity, we developed a model of PDAC tumor overexpressing interleukin-6 (IL-6), a known driver of pancreatic tumorigenesis and cancer cachexia, in orthotopically implanted PDAC cancer cells (OT-PDACIL6). This model utilized codon-optimized IL6 to accurately detect tumor cells in the pancreas and distal organs via qPCR. OT-PDACIL6 tumors developed poorly- differentiated, infiltrative carcinomas, which were histologically similar to the PDAC cell line they were derived from (OT-PDACparental). Surprisingly, OT-PDACIL6 tumors were present at 5 days post implantation, but undetectable by histology or qPCR by 10 days post implantation. OT- PDACIL6 and sham mouse survival were equivalent (87.5%) at 76 days post-implantation. OT- PDACparental mortality was 100% at day 13. We concluded that OT-PDACIL6 tumors resolve spontaneously. IL-6 is well-established as a driver of cachexia, and we saw that OT-PDACIL6 tumors induced a rapid cachexia phenotype that began to resolve as the tumor was cleared. Plasma IL-6 levels tracked with tumor burden. At 5 days, plasma IL-6 was 100-fold higher in OT-PDACIL6 mice than in OT-PDACparental mice, but undetectable in OT-PDACIL6 mice after 12 days. We hypothesized that high levels of IL-6 were driving an anti-tumor immune response. To assess changes in tumor immune infiltrate that could lead to tumor clearance, we measured a broad range of immune cells using flow cytometry and immunofluorescent histology. At peak tumor burden, we observed exacerbated splenomegaly, and increased tumor-infiltrating T cells and NK cells in OT-PDACIL6 mice. T regulatory cells were decreased in OT-PDACIL6 tumors. CD4/CD8 antibody-based depletion prevented tumor clearance and significantly decreased survival of OT-PDACIL6 mice, indicating that T cells are necessary for IL-6-driven PDAC tumor clearance. We then tested whether the anti-tumor T cell response was durable using a secondary tumor challenge with PDACparental cells. This revealed that the anti-tumor T cell response elicited by mice bearing OT-PDACIL6 tumors was sufficient to prevent development of OT-PDACparental tumors, likely due to induction of long-term memory T cells during the initial tumor challenge. We assessed the effect of the anti-tumor immune response on metastases and found that PDACIL6 cells were detected by qPCR in lungs of 58% of mice at day 5, and not detected at day 12. We conclude that extremely high levels of tumor-derived IL-6 are sufficient to drive an anti-tumor T cell response that rapidly and durably clears the tumor. The combination of increased tumor-infiltrating T cells and decreased T regulatory cells are likely both important aspects of the immune response observed in OT-PDACIL6 mice. This surprising finding highlights the pleiotropic effects of IL-6, which is known to promote tumorigenesis and cachexia, and yet, in this work, is the driving force underlying tumor clearance. Citation Format: Paige C Arneson-Wissink, Alexandra Q Bartlett, Heike Mendez, Xinxia Zhu, Jessica Dickie, Matthew McWhorter, Peter R Levasseur, Parham Diba, Katelyn T Byrne, Gregory D Scott, Robert Eil, Aaron J Grossberg. Durable T-cell-mediated anti-tumor immune response to pancreatic cancer cells overexpressing interleukin 6 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B044.

Cell adhesion genes expression level in oropharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma: a preliminary study

Head and neck cancers (HNCs) are tumors developed in the upper aerodigestive tracts, more than 90% originating in the squamous cells lining the mucosa of the upper airways. There are between 650,000 and 900,000 new cases each year, most of them advanced, resulting in high mortality (five-year survival about 50%). HNCs do not metastasize to distant sites, but often affect local lymph nodes. Their etiology is multifactorial, with smoking, alcohol consumption and HPV infection being the most common. HNCs rapidly enter hypoxia and synthesize angiogenic factors, which contribute to further tumor development and recurrence. Although there is a relatively wide variety of therapeutic strategies, their effectiveness is reduced because of resistance development. Cell adhesion mediated by integrins and the extracellular matrix is an important mechanism of tumor progression. In this study, expression levels of genes involved in cell adhesion were determined in two patients with oropharyngeal squamous cell carcinoma and three patients with laryngeal squamous cell carcinoma. The obtained data showed that there is a differentiation in the expression of genes for collagens (COL15A1, COL18A1, COL4A1, COL4A2 and COL4A3), integrins (ITGA4, ITGAV, ITGB3) and two molecules involved in their interactions (CD44 and MMP2) for the two tumor types, the results obtained need validation on a statistically significant sample.

The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Tumors Established in a Defective Immune Environment Reprogram the Oncogenic Signaling Pathways to Escalate Tumor Antigenicity.

Tumors developed in immunocompromised hosts are more immunogenic. However, few studies have addressed the potential mechanisms underlying the high immunogenicity of tumors found in a suppressed immune system. Therefore, we aimed to elucidate the impacts of the immune system on tumor behaviors and immunogenicity sculpting. A murine colorectal adenocarcinoma cell line, CT26wt, was administrated into immunocompetent (BALB/c) and immunocompromised (NOD.SCID) mice, respectively. On day 11, the CT26 cells slowly progressed in the NOD.SCID mice compared to the BALB/c mice. We then performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) and analyzed the differentially expressed proteins (DEPs). The DEPs participated in numerous oncogenic pathways, PI3K/AKT/mTOR cell signaling, and the silencing of several tumor suppressors, such as PTEN and RBL1, during tumorigenesis. On day 34, the CT26/SCID tumors inversely became malignant counterparts; then the CT26/SCID tumors were harvested and re-inoculated into immunocompetent mice (CT26/SCID-Re tumors) to determine the immunogenicity. The CT26/SCID-Re tumor growth rate significantly decreased. Furthermore, increased infiltrations of dendritic cells and tumor-infiltrating T lymphocytes were found in the CT26/SCID-Re tumors. These findings suggest that immunogenic tumors might express multiple tumor rejection antigens, unlike wild-type tumors, and attract more immune cells, therefore decreasing the growth rate. Collectively, our study first revealed that in immunodeficient hosts, tumor suppressors were silenced and oncogenic signaling pathways were changed during the initial phase of tumor development. With tumor progression, the tumor antigens were overexpressed, exhibiting elevated immunogenicity. This study offers a hint on the mechanisms of tumorigenesis and provides a niche for investigating the interaction between host immunity and cancer development.

Abstract 119: A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer

Abstract Recapitulating physiologically relevant tumor microenvironment is important yet a significant challenge in modeling colorectal cancer (CRC) for basic and translational studies. Among the different methods, using the cecal-wall injection technique is the most common way to create animal models of colorectal cancer (CRC). However, this approach requires complex surgery, and its unnatural placement creates a significant obstacle for developing treatments. Here, we report a non-surgical procedure for establishing orthotopic CRC mouse models, reflecting the appropriate tumor microenvironment to better model the disease development and facilitate translational studies including drug identification and validation. In this procedure, anesthesia is induced in overnight fasted study immunocompetent or immunodeficient mice while placed on a thermostat-controlled heating blanket. A specialized catheter is inserted and distended into the mouse colon to gently block the colon and ensure the localization for tumor development. Hereafter, the colonic epithelium is treated with proteases and mechanical abrasion to cause mild disruption. Following the local gut inflammation, the targeted colon region is incubated with tumor cells, leading to the adhesion of tumor cells to the colonic epithelium. Based on the animal background and the cell line used, the tumor developed within 3-4 weeks. The stages of tumor development at different time points were successfully confirmed using hematoxylin and eosin stain. Tumor microenvironment data revealed a significant increase in the immune cell infiltration specifically CD4+ T-cells and tumor-associated macrophages in the non-surgical procedure as compared to the cecal-wall injection. Furthermore, a significant increase in the PD-1 expression of the CD8+ T-cells in the cecal-wall injection as compared to the non-surgical procedure, suggests that the precise location of the tumor site may exert a substantial influence on pre-clinical therapeutic outcomes of immune-checkpoint inhibitors, thereby potentially hampering the clinical efficacy of drug treatments. Additional adaptations of this procedure may include: i) tumor development in different mouse strains, ii) local delivery of adeno-Cre in transgenic mice for CRC, and iii) delivering tumor organoids and patient-derived xenografts in immuno-deficient mice. Overall, our non-surgical procedure overcomes the technical difficulties of surgery and offers several advantages like ease of handling, cost-effectiveness, pain-free with nearly no procedure-associated mortality and adaptability to several strains. In conclusion, this procedure provides novel and promising alternative to conventional surgical orthotopic CRC mouse models for therapeutic interventions. Citation Format: Naresh Singh, Samantha Sharma, Kevin Van Der Jeught, Zhuolong Zhou, Xinna Zhang, Xiongbin Lu. A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 119.

Abstract 6758: Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa

Abstract Activation of the stimulator of interferon genes (STING) pathway causes the release of factors that trigger the immune response in the tumor microenvironment (TME). Transient strong activation leads to immune activation and tumor suppression, while prolonged weak activation leads to immuno-suppression promoting tumor development. Reprogramming of M2-type tumor-associated macrophages (M2-TAMs) toward an M1 phenotype, suppression of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (mMDSCs), as well as activation of dendritic cells (DCs) to train T-cells are advantageous, while the activation of STING pathway in T-cells is leads to their apoptosis. Thus, targeted delivery of STINGa to specific cells with TME to induce strong transient activation may be a key to therapy. We have developed a delivery approach that uses one or two pH Low Insertion Peptides (pHLIPs) that collaborate in the targeted intracellular delivery of a STINGa. STINGa were conjugated with pHLIPs via S-S cleavable self-immolating linkers. Biophysical studies were performed to ensure proper interactions of pHLIP-STINGa agents with membrane lipid bilayers. pHLIPs extend the lifetime of STINGa in the blood and target them to acidic cancer and stromal cells (CAFs), as well as M2-TAMs, mMDSCs and DCs within TME. The targeting results in selective cytokine activation in tumors with no systemic activation, which triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after one or two doses of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. No tumors developed in mice re-challenged by an additional injection of cancer cells in a 90-day experiment. Thus, targeted delivery of STINGa to cancer cells, tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T- and NK-cells, which gain access to the tumor core. The cytotoxic activity of T- and NK-cells is not impaired by the acidic environment and immune memory is developed. The pHLIP technology may allow transformation of immuno-activating agents into more potent therapeutics, since pHLIP can target and deliver these agents to cancer cells, tumor stroma and myeloid cells. Citation Format: Michael DuPont, Anna Moshnikova, Marissa Iraca, Craig Klumpp, Hannah Visca, Dana Allababidi, Phoebe Pelzer, Donald Engelman, Oleg Andreev, Yana Reshetnyak. Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6758.

Abstract 1451: Replication repair deficient mouse models provide insights into gliomagenesis and response to immunotherapy

Abstract Background: Replication Repair Deficiency (RRD), caused by germline monoallelic (Lynch Syndrome) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes, is present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD glioblastomas are chemoradiation-resistant, but respond favorably to immune checkpoint inhibition (ICI). Representative immunocompetent animal models are urgently needed for 3 recently identified subgroups based on specific somatically-acquired mutations, survival, and immunotherapy response (RRD1: MMRD with POLE mutations, RRD2: MMRD associated with TP53 mutations, and RRD3: MMRD harboring IDH1 mutations). Methods: Using germline mutations and brain-specific Cre-drivers, we genetically engineered mouse models that recapitulate each human RRD-subgroup. Results: All mouse models robustly developed brain tumors displaying phenotypic variation. RRD1 (Nestin- and Olig2-Cre+/Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): CMMRD-like Nestin-Cre-driven mice develop posterior-fossa glioma-like or embryonal (EB)-like tumors at ~2.7 months. Olig2-Cre-driven mice display hemispheric gliomas at ~10 months, suggesting distinct cell-of-origin. RRD2 (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1−/−): tumors develop primarily in the hindbrain in germline Mlh1 mice, and majority arise in the forebrain of Nestin-Cre/Msh2 mice, highlighting the timeline of mutagenesis. Strikingly, germline Mlh1 tumors occur earlier than Nestin-Cre-driven RRD2 tumors, indicating early developmental mutation accumulation in CMMRD-patients. Lynch-like RRD1/2 mice succumb exclusively to gliomas &amp;gt;13 months (p&amp;lt;0.0001). RRD3 (Olig2-Cre+/Msh2LoxP/LoxP/Trp53LoxP/LoxP/LSL-Idh1R132H/+): mice succumb to brain tumors at &amp;gt;11 months and are hemispheric. Significance: These observations recapitulate human data, where CMMRD-patients develop glioblastoma/EB earlier than Lynch-patients (8.6 vs. 14-years; p&amp;lt;0.0001), and posterior-fossa glioblastoma/EB presents earlier than hemispheric gliomas (p=0.04). Additionally, tumor onset and location vary by subgroup (RRD1: 7.6-years, RRD2: 8.3-years, both hemispheric/posterior-fossa; RRD3: 12-years, hemispheric; p=0.005). In both mice and humans, RRD1 exhibits ultra-hypermutation, high immune infiltration, and response to ICI, whereas RRD2 harbors lower mutational burden, are immune-cold, and ICI-monotherapy resistant. Temporal dynamics of RRD tumor development is currently being tracked by serial MRI to define previously undetermined biologically relevant time points of tumor progression. Conclusion: Our models accurately mimic the human condition and provide unique insights into RRD tumorigenesis, allowing optimization of subgroup-tailored therapeutic approaches. Citation Format: Zoya Aamir, Melissa Galati, Emma Gattoni, Owen Crump, Nuno M. Nunes, Anirban Das, Nicholas Fernandez, Nicholas Fernandez, Angel K. Wong, Jerome Fortin, Lucie Stengs, Vanessa Bianchi, Melissa Edwards, Logine Negm, Cynthia Hawkins, David Malkin, Sean Egan, Uri Tabori. Replication repair deficient mouse models provide insights into gliomagenesis and response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1451.

Clonal analysis of metachronous double biliary tract cancers.

The molecular mechanisms underpinning the development of metachronous tumors in the remnant bile duct following surgical resection of primary biliary tract carcinomas (BTCs) are unknown. This study aimed to elucidate these mechanisms by evaluating the clinicopathologic features of BTCs, the alterations to 31 BTC-related genes on targeted sequencing, and the aberrant expression of p53, p16, SMAD4, ARID1A and β-catenin on immunohistochemistry. Twelve consecutive patients who underwent resection of metachronous BTCs following primary BTC resection with negative bile duct margins were enrolled. Among the 12 metachronous tumors, six exhibited anterograde growth in the lower portion and six exhibited retrograde growth in the upper portion of the biliary tree. Surgical resection of metachronous BTCs resulted in recurrence-free survival in seven, local recurrence in five, and death in two patients. Nine achieved 5-year overall survival after primary surgery. Molecular analyses revealed that recurrently altered genes were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D and ERBB3. Each of these was altered in at least three cases. A comparison of the molecular features between 12 paired primary and metachronous BTCs indicated that 10 (83%) metachronous tumors developed in clonal association with corresponding primary tumors either successionally or phylogenically. The remaining two (17%) developed distinctly. The successional tumors consisted of direct or evolved primary tumor clones that spread along the bile duct. The phylogenic tumors consisted of genetically unstable clones and conferred a poor prognosis. Metachronous tumors distinct from their primaries harbored fewer mutations than successional and phylogenic tumors. In conclusion, over 80% of metachronous BTCs that develop following primary BTC resection are probably molecularly associated with their primaries in either a successional or a phylogenetic manner. Comparison between the molecular features of a metachronous tumor and those of a preceding tumor may provide effective therapeutic clues for the treatment of metachronous BTC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Evaluation of the prognostic significance of clinical features of tumoral lesions in an extensive series of mycosis fungoides.

Tumors indicating the advanced stage of mycosis fungoides (MF) have a rich clinical spectrum. Although it is known that the prognosis of MF generally worsens following the development of tumors, some cases may have a relatively indolent course, and the role of clinical characteristics regarding prognosis has still not been well understood. MF patients were retrospectively evaluated regarding the development of tumors. Besides demographic characteristics, data of the subtype and stage of the disease were recorded. The clinical features of tumors, including number (<5, 5-10, 11-20, or >20), location, dimension (diameter of ≥5 cm), presence of ulceration, and surrounding inflammation, were noted. Univariate and multivariate analyses evaluated the relationship between overall survival (OS) with demographic and clinical features. Among 730 consecutive MF patients, tumors developed in 8.2% (n = 60), of whom 46.7% were diagnosed with advanced-stage MF from the beginning. The most common subtype was folliculotropic MF (53.3%). Most patients (55%) had multiple tumors, and the most frequent localization was the trunk (71.7%). Most tumors presented as smooth-surfaced, indurated papules and/or nodules (70%), while others were reddish-purple, occasionally accompanied by ulceration (50%), perilesional inflammation (23.3%), and attaining large dimensions (25%). Mortality was recorded in 51.7% of patients, and the 5-year OS rate from the diagnosis of tumors was 49%. Independent poor prognostic factors for OS in multivariate analysis included older age at the time of diagnosis, presence of tumors at the initial MF diagnosis, presence of over 20 tumors, and the existence of large tumors. Tumoral MF seen in older patients, the first diagnosis of MF in this stage, presenting with generalized and large tumors, seems to be a predictive factor for OS.

Investigation of Clinical, Laboratory, Imaging Findings and Histopathological Features of Patients wıth Gastric Neuroendocrine Cell Hyperplasia.

Neuroendocrine cell hyperplasia is a non-neoplastic proliferation of enterochromaffin-like cells and is considered a premalignant lesion because of their potential to progress to neuroendocrine tumor. In this study, we aimed to evaluate the demographic and clinical features, laboratory, radiological and endoscopic findings, gastric biopsy histopathological features, follow-up frequency, and histopathological findings of patients diagnosed with gastric neuroendocrine cell hyperplasia as well as to investigate the factors that play a role in the development of neuroendocrine tumors on the basis of neuroendocrine cell hyperplasia. The study has been conducted in 2 centers with 282 patients that were grouped as those with and without neuroendocrine tumor. Individuals with control endoscopy were separated as those with regression of neuroendocrine cell hyperplasia and those without regression, and the determined parameters were evaluated between the groups. The most common histological subtype of neuroendocrine cell hyperplasia was linear+micronodular (50.4%). Neuroendocrine tumor developed in 4.3% (12/282) of the patients with neuroendocrine cell hyperplasia after a mean of 36 months. The presence of polyps as confirmed via endoscopy and dysplasia as confirmed via histopathological examination was significantly higher in favor of the group with neuroendocrine tumor (P = .01). In patients with neuroendocrine cell hyperplasia regressed and patients in whom it did not regress were examined, the rate of asymptomatic patients and increased sedimentation rate were found in favor of the group that did not regress (P = .02 and P = .02), but no difference was found in other parameters. Neuroendocrine tumor development rate was found to be 4.3% in the background of neuroendocrine cell hyperplasia. Two factors predicting progression from neuroendocrine cell hyperplasia to neuroendocrine tumor can be elaborated as the presence of polypoid appearance due to neuroendocrine cell hyperplasia as confirmed via endoscopy and dysplasia as confirmed via histopathological examination.

Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

The role of non-invasive preoperative imaging techniques in predicting the risk of pancreatic fistula development in pancreaticoduodenal tumours

Background. In pancreaticoduodenal tumours, pancreaticoduodenectomy (PDE) is a radical surgical intervention. The most important stage of the operation is the pancreaticojejunoanastomosis (PEA), the admissibility of which depends on many factors: the degree of fibrous changes in the pancreatic parenchyma, the degree of mechanical jaundice, and the operating surgeon’s technique. Identification of factors that reflect changes in the structure of the pancreatic parenchyma and its ductal system, which are important predictors in predicting the risk of developing pancreatic fistula (PF) after PDE, is of great scientific importance. Purpose. Evaluation of changes in the structure of the pancreatic parenchyma and its ductal system using non-invasive preoperative imaging methods in predicting the risk of pancreatic fistula development during pancreaticoduodenectomy. Materials and Methods. This study included 302 patients who underwent pancreaticoduodenectomy. Mechanical jaundice was detected in 246 (81.5%) patients, 56 (18.5%) patients had no jaundice, the age of patients ranged from 31 to 77 years, 178 (58.9%) were men and 124 (41.1%) were women. Ultrasound elastography and multidetector computed tomography were used for preoperative non-invasive visualisation of the degree of changes in the pancreatic parenchyma and its ductal system. The following parameters of visualisation of the pancreatic parenchyma with an assessment of the anatomical features of the isthmus and the duct of Wirsung were identified: diameter and cross-sectional area of the duct of Wirsung (at the level of the isthmus), width and thickness of the pancreas (at the level of the isthmus), cross-sectional area of the pancreatic isthmus (without the area of the duct of Wirsung), native density, and stiffness of the pancreatic parenchyma. Results and discussion. The ROC analysis of the parameters for assessing the state of the parenchyma of the pancreas and the duct of Wirsung obtained from computed tomography made it possible to determine the validity of these indicators in predicting the risk of developing PF. According to the results of the ROC analysis, the following indicators were identified as markers of moderate risk of developing PF: the diameter of the duct of Wirsung is less than 4 mm, the cross-sectional area of the duct of Wirsung is less than 0.23 cm2 , the thickness of the isthmus of the pancreas is greater than 15 mm, the width of the isthmus of the pancreas is greater than 19 mm, the cross-sectional area of the isthmus of the pancreas (without the area of the duct of Wirsung) is greater than 3 cm2 , and the native density is greater than 24 HU. To assess the high risk of developing PF, the use of ROC analysis allowed us to establish the high quality of diagnostic models for such computed tomography parameters as the diameter of the duct of Wirsung – the area under the ROC curve is 0.965, the cross-sectional area of the duct of Wirsung – AUC is 0.894, the cross-sectional area of the isthmus of the pancreas (without the area of the duct of Wirsung) – AUC is 0.873, the width of the isthmus of the pancreas – the area under the ROC curve is 0.859, the native density – AUC is 0.844. Determination of the parameter of stiffness of the pancreatic parenchyma by shear wave elastography in predicting high and moderate risk of developing PF also corresponded to the high quality of the diagnostic model. Conclusions. The use of non-invasive imaging methods allows changes in the structure of the pancreatic parenchyma, anatomical features of the pancreatic isthmus and the duct of Wirsung to be detected at the preoperative stage, the risk of developing a pancreatic fistula to be predicted and the number of complications during pancreaticoduodenectomy to be reduced.

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma.

Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC. Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)-knockdown and -overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7. The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC. This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Abstract B039: Effect of cross-sex testosterone therapy on Pik3ca-driven breast cancer incidence in mice

Abstract Background: The majority of transmasculine individuals—assigned female at birth—pursue gender-affirming testosterone therapy (TT) to treat their gender dysphoria. It remains unknown the extent to which TT modulates the breast, including breast cancer (BC) risk, in transmasculine individuals. Given that hormone regulation of breast development is similar in mice and humans, mouse models are an excellent methodology to understand the effect of TT on BC development. We used a BC mouse model, MMTV-Cre Pik3caf/wt, to investigate the effect of testosterone on estrogen receptor positive (ER+) BC development. Method: Immunohistochemistry confirmed that our MMTV-Cre Pik3caf/wt tumors are ER+. We established 5 study arms: female controls (n=4), oophorectomized female controls (n=6), male mice (n=5), female mice receiving weekly 400 µg s.c. of testosterone cypionate (n=5), and oophorectomized female mice receiving weekly testosterone cypionate (n=5). Mammary tumor development was monitored weekly; palpable tumors were measured 3x a week. Mice were euthanized when their largest tumor reached 1cm, or if multiple tumors arise, when the combined tumor burden reaches 2 cm. Mice that have not reached endpoint by 48 weeks of age were euthanized and censored. The total number of tumors were observed during necropsy. Results: All 4 female controls developed a mean of 4 tumors each and were euthanized at around 35 weeks as the largest tumor reached 1 cm. The remaining mice were euthanized study endpoint of 48 weeks. Only 1 of 5 female mice receiving testosterone had a macroscopic tumor at 48 weeks. Hence, the total number of tumors developed among female mice receiving testosterone are similar or lower than female controls. Three out of 6 of oophorectomized females developed an average of 2 tumors while 2 of 5 female oophorectomized mice receiving testosterone developed 3 tumors each. Thus, when compared to intact females, oophorectomy appears to increase the lag time to Pik3ca tumor development. Lastly, 1 of 5 male mice developed 4 tumors. Conclusion: This pilot study demonstrated that testosterone does not increase ER+ BC incidence in Pik3ca-transgenic mice. Findings in oophorectomized mice suggest that aromatization of testosterone to estradiol may not contribute significantly to tumor development in these mice. Our findings provides insights into about BC risk associated with TT in transmasculine individuals. More studies are warranted to understand the health effects of gender-affirming hormone therapy, and reduce healthcare disparities in the transgender community. Citation Format: Lin Wang, Abhishek Thavamani, Erica S. Massicott, Vanessa C. Bret-Mounet, Joseph Abirached, John G. Clohessy, Gabrielle M. Baker, Gerburg M. Wulf, Yujing Jan Heng. Effect of cross-sex testosterone therapy on Pik3ca-driven breast cancer incidence in mice [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B039.

Digital twins for breast cancer treatment – an empirical study on stakeholders’ perspectives on potentials and challenges

PurposeWith 2.3 million diagnoses and 685,000 deaths annually, breast cancer is the most common cancer in women. The provision of necessary information throughout the whole patient journey is key to minimize the risk of breast cancer, to detect breast cancer as early as possible, and to aid the treatment process. Digital solutions provide abilities to holistically collect, transfer, and sophisticatedly analyze information. Specifically, digital twins in healthcare, as dynamic replicas of human bodies, are promising approaches for monitoring the condition of their patients and predicting tumor developments based on biometric data. However, the acceptance and adoption of such digital twin solutions in healthcare heavily depend on the individual stakeholders of the treatment process. This study aims to identify potentials and challenges of the introduction of digital twins in breast cancer applications from the involved stakeholders’ perspectives.MethodsWe conducted semi-structured interviews with 14 relevant stakeholders from the breast cancer treatment process. The interviews were then analyzed, based on the qualitative content analysis according to Mayring.ResultsThe results show that stakeholders see great potential in digital twin solutions to further facilitate personalized medicine, efficiency increases, and scientific benefits. However, the sensitive nature of healthcare causes numerous potential challenges in the technical, regulatory, user interface, and the strategic domain.ConclusionsThe stakeholders unanimously agreed on the potential benefits of digital twins. However, existing systemic and individual stakeholder-level barriers hamper their introduction in breast cancer settings.

A phenol amine molecule from Salinivenus iranica acts as the inhibitor of cancer stem cells in breast cancer cell lines

In recent years, the anticancer properties of metabolites from halophilic microorganisms have received a lot of attention. Twenty-nine halophilic bacterial strains were selected from a culture collection to test the effects of their supernatant metabolites on stem cell-like properties of six human cancer cell lines. Human fibroblasts were used as normal control. Sphere and colony formation assay were done to assess the stem cell-like properties. invasion and migration assay, and tumor development in mice model were done to assess the anti-tumorigenesis effect in vitro and in vivo. The metabolites from Salinivenus iranica demonstrated the most potent cytotoxic effect on breast cancer cell lines (IC50 = 100 µg/mL) among all strains, with no effect on normal cells. In MDA-MB-231 cells, the supernatant metabolites enhanced both early and late apoptosis (approximately 9.5% and 48.8%, respectively) and decreased the sphere and colony formation ability of breast cancer cells. Furthermore, after intratumor injection of metabolites, tumors developed in the mice models reduced dramatically, associated with increased pro-apoptotic caspase-3 expression. The purified cytotoxic molecule, a phenol amine with a molecular weight of 1961.73 Dalton (IC50 = 1 µg/mL), downregulated pluripotency gene SRY-Box Transcription Factor 2 (SOX-2) expression in breast cancer cells which is associated with resistance to conventional anticancer treatment. In conclusion, we suggested that the phenol amine molecule from Salinivenus iranica could be a potential anti-breast cancer component.

Acrolein produced by glioma cells under hypoxia inhibits neutrophil AKT activity and suppresses anti-tumoral activities

Acrolein, which is the most reactive aldehyde, is a byproduct of lipid peroxidation in a hypoxic environment. Acrolein has been shown to form acrolein-cysteine bonds, resulting in functional changes in proteins and immune effector cell suppression. Neutrophils are the most abundant immune effector cells in circulation in humans. In the tumor microenvironment, proinflammatory tumor-associated neutrophils (TANs), which are termed N1 neutrophils, exert antitumor effects via the secretion of cytokines, while anti-inflammatory neutrophils (N2 neutrophils) support tumor growth. Glioma is characterized by significant tissue hypoxia, immune cell infiltration, and a highly immunosuppressive microenvironment. In glioma, neutrophils exert antitumor effects early in tumor development but gradually shift to a tumor-supporting role as the tumor develops. However, the mechanism of this anti-to protumoral switch in TANs remains unclear. In this study, we found that the production of acrolein in glioma cells under hypoxic conditions inhibited neutrophil activation and induced an anti-inflammatory phenotype by directly reacting with Cys310 of AKT and inhibiting AKT activity. A higher percentage of cells expressing acrolein adducts in tumor tissue are associated with poorer prognosis in glioblastoma patients. Furthermore, high-grade glioma patients have increased serum acrolein levels and impaired neutrophil functions. These results suggest that acrolein suppresses neutrophil function and contributes to the switch in the neutrophil phenotype in glioma.

Impact of Simulated Rotating Shift Work on Mammary Tumor Development in the p53R270H©/+WAPCre Mouse Model for Breast Cancer.

Epidemiological studies associate night shift work with increased breast cancer risk. However, the underlying mechanisms are not clearly understood. To better understand these mechanisms, animal models that mimic the human situation of different aspects of shift work are needed. In this study, we used "timed sleep restriction" (TSR) cages to simulate clockwise and counterclockwise rotating shift work schedules and investigated predicted sleep patterns and mammary tumor development in breast tumor-prone female p53R270H©/+WAPCre mice. We show that TSR cages are effective in disturbing normal activity and estimated sleep patterns. Although circadian rhythms were not shifted, we observed effects of the rotating schedules on sleep timing and sleep duration. Sleep loss during a simulated shift was partly compensated after the shift and also partly during the free days. No effects were observed on body weight gain and latency time of breast cancer development. In summary, our study shows that the TSR cages can be used to model shift work in mice and affect patterns of activity and sleep. The effect of disturbing sleep patterns on carcinogenesis needs to be further investigated.