Tumor Tissue Sections Research Articles

Abstract 3745: Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression

Abstract Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.

Measurement of biological washout rates depending on tumor vascular status in 15O in-beam rat-PET

Objective. The biological washout of positron emitters should be modeled and corrected in order to achieve quantitative dose range verification in charged particle therapy based on positron emission tomography (PET). This biological washout effect is affected by physiological environmental conditions such as blood perfusion and metabolism, but the correlation to tumour pathology has not been studied yet. Approach. The aim of this study was to investigate the dependence of the biological washout rate on tumour vascular status in rat irradiation. Two types of tumour vascularity conditions, perfused and hypoxic, were modelled with nude rats. The rats were irradiated by a radioactive 15O ion beam and time activity curves were acquired by dynamic in-beam PET measurement. Tumour tissue sections were obtained to observe the histology as well. The biological washout rate was derived using a single-compartment model with two decay components (medium decay, k 2m and slow decay, k 2s ). Main results. All k 2m values in the vascular perfused tumour tissue were higher than the values of the normal tissue. All k 2m values in the hypoxic tumour tissue were much lower than the values of the vascular perfused tumour tissue and slightly lower than the values of the normal tissue. Significance. The dependency of the biological washout on the tumour vasculature conditions was experimentally shown.

A derivant of ginsenoside CK and its inhibitory effect on hepatocellular carcinoma

Epidemiological studies have shown that hepatocellular carcinoma (HCC) is a main cause of tumor death worldwide. Accumulating data indicate that ginsenoside CK is an effective compound for preventing HCC growth and development. However, improvement of pharmaceutical effect of the ginsenoside CK is still needed. In our study, we performed acetylation of ginsenoside CK (CK-3) and investigated the antitumor effects of the derivative in vitro and in vivo. The cytotoxicity analysis revealed that compared with CK, CK-3 could inhibit the proliferation of multiple tumor cell lines at a lower concentration. Treating with CK-3 on HCC cells arrested cell cycle in G2/M phase and induced cell apoptosis through AO/EB staining, TUNEL analysis and flow cytometry. Meanwhile, CK-3 significantly inhibited tumor growth in an HCC xenograft model and showed no side effect on the function of the main organs. Mechanistically, whole transcriptome analysis revealed that the antitumor effect of CK-3 was involved in the Hippo signaling pathway. The immunoblotting and immunofluorescence results illustrated that CK-3 directly facilitated the phosphorylation of YAP1 and decreased the expression of the main transcription factor TEAD2 in HCC cell lines and tumor tissue sections. Collectively, our results demostrate the formation of a new derivative of ginsenoside CK and its regulatory mechanism in HCC, which could activate the Hippo-YAP1-TEAD2 signaling pathway to regulate HCC progression. This research could provide a new direction for traditional Chinese medicine in the therapy of tumors.

Predictive significance of tumor-associated miRNA-21 and -221 expression in patients with prostate cancer.

e17015 Background: It is thought that the occurrence and progression of tumors, such as prostate cancer (PCа), may be due to decreased levels of tumor-inhibiting microRNAs and/or overexpression of oncogenic microRNAs, but their specificity and role in tumor progression are contradictory. That is why the study of the possibility of using miRNAs as prognostic markers is especially relevant. Objective: to investigate the features of tumor -associated microRNAs-21 and -221 expression in PCa tissue with different risk of progression and to determine the relationship of their indicators with the molecular profile of tumors. Methods: The study was conducted on the clinical material of 60 patients with PCa stage II-III, who were treated in Rivne Regional Hospital and Rivne oncological center during 2018-2021 years. Determination of the risk of PCa progression was performed by D'Amico et al. (1998). Immunohistochemical study of Ki-67 expression (clone MIB -1) and androgen receptors (AR) (clone 441) were performed on paraffin sections of tumor tissue using the Ultra Vision LP Detection System (Thermo Scientific, USA). Determination of microRNA-21 and -221 levels were determined using real-time quantitative PCR. Statistical processing of the results was performed using GraphPad Prism 8. Results: It is determined that a characteristic feature of PCa with a low risk of progression by D'Amico et al . are low expression of oncogenic miRNA-21 and -221 in tumor tissue. In particular, in the PCa samples of high risk of progression (n = 30) the level of miRNA-21 was 3.2 times (p < 0.05), and miRNA-221 was 1.35 times (p < 0.05) higher compared to tumor tissue of patients with low progression (n = 30). The existence of a direct correlation between the expression of miRNA-21 and the proliferative activity of PCa (ρ = 0.63; p < 0.05) is shown. We found that the expression of miRNA-221 is associated with a high level of AR expression (ρ = 0.43; p < 0.05) . Conclusions: The obtained data indicate the relationship of tumor -associated microRNA-21 and -221 with the risk of tumor progression and such molecular biological characteristics of tumors as proliferative activity and receptor status, which indicates the prospects for their further study as additional markers for predicting of PCa course.

A multiplexed immunohistochemical consecutive staining on single slide (MICSSS) analysis of the immune microenvironment of bile duct cancers (BDC) pre and post neoadjuvantchemotherapy (NACT).

e16151 Background: NACT is increasingly being used in the management of locally advanced BTC. Emerging evidence suggests a potential key contributing role of tumor infiltrating immune cells in the prognosis & response to therapy. We set out to characterize immune modulation of tumor immune microenvironment composition in BTC following NACT. Methods: Patients (pts) with locally advanced BTC who underwent a diagnostic biopsy, then NACT followed by resection between 2014 & 2018 were identified & consented after IRB approval. MICSSS, a sample-sparing chromogenic consecutive multiplex tissue staining method, was performed with a series of immune markers (Table), to characterize T cell subsets, B cells, macrophages, mature dendritic cells (DCs), and immune checkpoints on pre & post NACT formalin-fixed paraffin-embedded tumor tissue sections. Density was calculated for each marker (+ve cells/mm2) following annotation of tissues by tumor, fibrosis, necrosis, stromal & tumor infiltrating lymphocyte-enriched areas. Results: Nine pts were enrolled. Final analysis included 5 pts with adequate tissue. Median age = 48 (41-56), with 4 female, 4 intrahepatic cholangiocarcinomas & 1 gallbladder. All pts received Gemcitabine/Cisplatin as NACT with a median of 5 (4-7) cycles. Median time from diagnosis to surgery was 4.3 (1.4-7.8) months & last cycle to surgery was 0.9 (0.6-1.5) month. All pts were MMR proficient, 1 Her2+ & 2 with FGFR2 amplification. NACT on average produced a depletion of all immune markers (Table). Given the small N, each pt was considered their own control & changes in mean cell densities post NACT were calculated. Pt2 with a 40-fold increase in PDL1 expression & 5-fold decrease in CD8:FOXP3 ratio notably had the shortest disease-free interval (DFI). Pt3 with the longest DFI had the largest increase in CD8:FOXP3 by about 8-fold combined with a decrease in PDL1. Conclusions: Preliminary results suggest NACT may modulate immune microenvironment despite overall immune cell depletion. Future studies should focus on strategies to expand immune modulation of the tumor microenvironment in BTC by NACT, including immune oncology agent priming prior to or after NACT.[Table: see text]

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

In situ imaging for tumor microbiome interactions via imaging mass cytometry on single‐cell level

Co-detection of multiplex cancer subtypes and bacteria subtypes in situ is crucial for understanding tumor microbiome interactions in tumor microenvironment. Current standard techniques such as immunohistochemical staining and immunofluorescence staining are limited for their multiplicity. Simultaneously visualizing detailed cell subtypes and bacteria distribution across the same pathological section remains a major technical challenge. Herein, we developed a rapid semi-quantitative method for in situ imaging of bacteria and multiplex cell phenotypes on the same solid tumor tissue sections. We designed a panel of antibody probes labeled with mass tags, namely prokaryotic and eukaryotic cell hybrid probes for in situ imaging (PEHPSI). For application demonstration, PEHPSI stained two bacteria subtypes (lipopolysaccharides (LPS) for Gram-negative bacteria and lipoteichoic acid (LTA) for Gram-positive bacteria) simultaneously with four types of immune cells (leukocytes, CD8 + T-cells, B-cells and macrophages) and four breast cancer subtypes (classified by a panel of 12 human proteins) on the same tissue section. We unveiled that breast cancer cells are commonly enriched with Gram-negative bacteria and almost absent of Gram-positive bacteria, regardless of the cancer subtypes (triple-negative breast cancer [TNBC], HER2+, Luminal A and Luminal B). Further analysis revealed that on the single-cell level, Gram-negative bacteria have a significant correlation with CD8 + T-cells only in HER2+ breast cancer, while PKCD, ER, PR and Ki67 are correlated with Gram-negative bacteria in the other three subtypes of breast cancers. On the cell population level, in TNBC, CD19 expression intensity is up-regulated by approximately 25% in bacteria-enriched cells, while for HER2+, Luminal A and Luminal B breast cancers, the intensity of biomarkers associated with the malignancy, metastasis and proliferation of cancer cells (PKCD, ISG15 and IFI6) is down-regulated by 29%-38%. The flexible and expandable PEHPSI system permits intuitive multiplex co-visualization of bacteria and mammalian cells, which facilitates future research on tumor microbiome and tumor pathogenesis.

Study on the chemotherapeutic effect and mechanism of cucurbitacin E on laryngeal cancer stem cells.

ObjectivesStudy on the chemotherapeutic effect and mechanism of cucurbitacin E (CuE) on laryngeal cancer stem cells.MethodsWe used flow cytometry to sort out CD133+ laryngeal cancer stem cells; trypan blue rejection assay to detect the survival rate of laryngeal cancer stem cells; Cell counting kit‐8 (CCK‐8) assay to detect the effect of CuE on the proliferation ability of stem cells and the chemotherapeutic potentiation of doxorubicin; Transwell assay to observe the effect of CuE on the migration ability of stem cells; and Western Blot to detect the effect of CuE on the expression level of stem cell‐associated proteins. The tumor volume of nude mice was measured at the end of the experiment, and paraffin sections of nude mice tumor tissues were prepared and stained with Hematoxylin and eosin (H&E). The expression of c‐MYC in tumor tissues of nude mice was further detected by immunohistochemistry, and the effect of CuE on the expression level of related proteins in tumor tissues of nude mice was detected by Western Blot.ResultsCuE reduced the survival rate, proliferation ability, and migration ability of laryngeal cancer stem cells in vitro, and that CuE had a chemotherapeutic potentiating effect on doxorubicin. The possible mechanism of the chemotherapeutic effect of CuE was to reduce the expression of c‐MYC protein, and the possible mechanism of chemotherapy synergy was to reduce the expression of ABCG2 and P‐gp protein.ConclusionCuE has a chemotherapeutic effect on laryngeal cancer stem cells, as well as a chemotherapy synergy.

Abstract P2-01-06: Detection of circulating tumor DNA post neoadjuvant chemotherapy using a personalized assay is associated with disease relapse

Abstract Background: Approximately 30% of patients with early breast cancer present disease relapse after surgery, and technologies that enable the detection of circulating tumor DNA (ctDNA) were shown to identify them earlier than standard imaging. In the present study, we aimed to interrogate the value of the SignateraTM personalized ctDNA assay for early detection of disease relapse. Methods: The study included 52 early breast cancer patients undergoing neoadjuvant chemotherapy from an ongoing translational, single-center study at Jules Bordet Institute, Brussels, Belgium. From each patient, primary tumor tissue sections, blood sample for normal DNA, and serial plasma samples were sent to NATERA Inc for ctDNA detection using the SignateraTM assay. Plasma samples were collected before the initiation of neoadjuvant chemotherapy (baseline), after neoadjuvant chemotherapy, before surgery (pre-surgery), and at follow-up post-surgery. Whole exome sequencing (WES) was performed on the primary tumor tissue and matched normal DNA to design a patient-specific ctDNA assay. The unique set of SNV’s identified in WES were tracked in serial plasma samples. Associations between baseline ctDNA detection and standard clinicopathological characteristics and primary tumor point mutations or copy number aberrations were evaluated. Association between ctDNA detection, pathological complete response (pCR) and event-free survival (EFS) were explored. Fisher, Mann-Whitney and Kruskal-Wallis tests were used to compare variables. Log-rank tests and Cox regressions were used for survival analyses. Hazard ratios (HRs) and confidence intervals (CIs) were obtained from Cox regressions. For multivariable tests, logistic regressions were used for binary outcomes and Cox regressions for survival outcomes. P-values were obtained by comparing models with and without the variable of interest using Chi-square test in ANOVAs. Results: Forty-four out of 52 (85%) initially selected patients had the required tumor cellularity (≥ 20%) and adequate tumor DNA quantity and quality and had personalized ctDNA assays designed. In these 44 patients, 154 plasma samples were successfully processed including 38, 41, 75, 38 at baseline, pre-surgery, follow-up (any timepoint) and last follow-up timepoint, respectively. Twenty (45%) patients had hormone receptor positive/HER2-negative, 13 (30%) triple-negative and 11 (25%) HER2-positive breast cancers. Detection of ctDNA was observed in 22 of 38 (58%) patients at baseline and was significantly associated with Ki67>20% and MYC copy number gain in the primary tumor. ctDNA detection at pre-surgery and at last follow-up was observed in 2 of 41 (5%) and 2 of 38 (5%) patients respectively. All patients who achieved pCR were ctDNA-negative at the pre-surgical time point. With a median follow-up of 3.03 years (range 0.39 - 5.85), we observed 8 relapses (7 distant and 1 local, respectively). After adjusting for pCR, ctDNA detection was associated with shorter EFS at pre-surgery and at last follow-up time points (HR: 53, 95% CI: 4.5-624, p<0.01, and HR: 31, 95% CI: 2.7-352, p<0.01, respectively), but not at baseline (HR: 1.4, 95% CI: 0.3-5.9, p=0.67). Conclusions: The detection of ctDNA post neoadjuvant chemotherapy is associated with disease relapse in early breast cancer supporting interventional trials in this setting. Citation Format: Elisa Agostinetto, Frédéric Cailleux, Matteo Lambertini, Françoise Rothé, Hsin-Ta Wu, Mustafa Balcioglu, Himanshu Sethi, Delphine Vincent, Giulia Viglietti, Andrea Gombos, Andreas Papagiannis, Isabelle Veys, Ahmad Awada, Christos Sotiriou, Ekaterina Kalashnikova, Alexey Aleshin, Denis Larsimont, David Venet, Michail Ignatiadis. Detection of circulating tumor DNA post neoadjuvant chemotherapy using a personalized assay is associated with disease relapse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-06.

The Mechanism of Human Umbilical Mesenchymal Stem Cells (HUMSC) on Biological Behavior of Human Papillomavirus (HPV) Cells Through Restraining the Programmed Cell Death Protein 1/Programmed Cell Death Ligand 1 (PD-1/PD-L1) Signal Pathway

Biological behavior of HPV cell was observed by HUMSC through restraining PD-1/PD-L1 signal pathway. And HUMSC was adopted as target cell for the treatment on HPV. The rat HPV model was established and divided into three groups including blank group, control group and test group according to different reagents being injected into rats. Use HE staining method to observe the cancerous transformation of tumor tissue sections. The gene presentation of PD-1/PD-L1 and lymphocyte was detected with Western blot. The invasion and migration condition of cancer cells was observed from experiment in vitro. The quantity of cancer cells in test group was the least. And invasion and migration ability in test group was the weakest. The control group was the second. The number of tumor cells in the blank group was the largest. Strongest ability to invade and migrate. The presentation of PD-L1 was restrained partly by HUMSC. The increasing of immune-associated cells could be prompted by HUMSC. The quantity of CD3+, CD4+ and CD8+ in PB was the most in test group. The expression of blank groups is the lowest than others restrained by HUMSC. And quantity of abundant immune cells including CD3+, CD4+ and CD8+ could be activated partly through activating immune action of body. And monitoring function of immune system on HPV cells could be increased effectively. The invasion and migration ability in vitro of HPV could be reduced partly.

PHD finger protein 20-like protein 1 (PHF20L1) in ovarian cancer: from its overexpression in tissue to its upregulation by the ascites microenvironment

BackgroundOvarian cancer is the most aggressive gynecological malignancy. Transcriptional regulators impact the tumor phenotype and, consequently, clinical progression and response to therapy. PHD finger protein 20-like protein 1 (PHF20L1) is a transcriptional regulator with several isoforms, and studies on its role in ovarian cancer are limited. We previously reported that PHF20L1 is expressed as a fucosylated protein in SKOV-3 cells stimulated with ascites from patients with ovarian cancer.MethodsWe decided to analyze the expression of PHF20L1 in ovarian cancer tissues, determine whether a correlation exists between PHF20L1 expression and patient clinical data, and analyze whether ascites can modulate the different isoforms of this protein. Ovarian cancer biopsies from 29 different patients were analyzed by immunohistochemistry, and the expression of the isoforms in ovarian cancer cells with or without exposure to the tumor microenvironment, i.e., the ascitic fluid, was determined by western blotting assays.ResultsImmunohistochemical results suggest that PHF20L1 exhibits increased expression in sections of tumor tissues from patients with ovarian cancer and that higher PHF20L1 expression correlates with shorter progression-free survival and shorter overall survival. Furthermore, western blotting assays determined that protein isoforms are differentially regulated in SKOV-3 cells in response to stimulation with ascites from patients with epithelial ovarian cancer.ConclusionThe results suggest that PHF20L1 could play a relevant role in ovarian cancer given that higher PHF20L1 protein expression is associated with lower overall patient survival.

Targeting Amyloids with [18F]AV-45 for Medullary Thyroid Carcinoma Positron Emission Tomography/Computed Tomography Imaging: A Pilot Clinical Study.

Medullary thyroid carcinoma (MTC) is a malignant neuroendocrine tumor with a high recurrence rate. Amyloid plaques formed from the misfolding of calcitonin are the key characteristics of MTC. Herein, we conducted a first-in-human pilot clinical study by applying a β-amyloid-specific radiotracer, [18F]AV-45, to positron emission tomography (PET)/computed tomography (CT) imaging of MTC. The presence of amyloid plaques in the tumor tissue sections from five MTC patients was confirmed by hematoxylin and eosin (H&E) and Congo Red staining. [18F]AV-45 selectively accumulated in the amyloid plaques in the continued tumor tissue sections with similar distribution patterns to the H&E and Congo Red staining. In addition, the [18F]AV-45 uptake can be largely blocked by its nonradioactive reference compound. The [18F]AV-45 accumulation in the thyroid, neck lymph nodes, and muscles in healthy human subjects is close to the background indicated by PET/CT imaging. In the comparison PET/CT imaging study of a recurrent MTC patient, 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) showed an elevated uptake by multiple neck lymph nodes. In contrast, only one of these neck lymph nodes had increased [18F]AV-45 uptake. Postoperative histopathological analysis confirmed the [18F]AV-45 PET-positive lymph node as MTC with amyloid deposition, while other [18F]FDG positive lymph nodes were free from MTC and amyloid plaques. Thus, [18F]AV-45 showed the promise for the clinical PET/CT imaging of MTC.

A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells’ surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.

Abstract P044: Real-time visualization of tumor cell phenotype and microenvironmental heterogeneity enabled by a hyperspectral fluorescence microendoscope

Abstract Tumor microenvironmental heterogeneity is a major driver of treatment resistance and variability in clinical response to therapy. This heterogeneity arises from variations in cellular phenotypes populating the tumor microenvironment (TME), their spatio-temporal localization and expression of surface markers, often associated with specific biological function – stemness, metabolism, proliferation, immune activation and others. Such features are usually studied through ex vivo immunofluorescence and cytometry to identify cellular phenotypes in TME; however, they cannot be applied in vivo for real time TME analysis. Moreover, current in vivo imaging techniques fail to provide real-time high-resolution visualization of the TME. Routinely employed biopsy tissue sampling through fine needle aspirates is limited by its invasive nature and inability to provide a global and dynamic overview of the TME, thus limiting our ability to study spatiotemporal TME dynamics and identify possible features leading to potentially resistant tumor phenotypes. In this study, we demonstrate the use of a hyperspectral fluorescence microendoscope (HFME) to monitor cellular phenotypes in TME with real-time visualization and high video imaging capability (~17 fps). Current fluorescence video microscopy is limited to simultaneous imaging of no more than 2 molecular markers with potential to be expanded to ~4 markers using dichroic mirrors and point detectors; the HFME demonstrated in this study can currently resolve 6 different molecular markers, simultaneously, using a multichannel linear array detector with potential to expand to 10 or more markers. Using a cocktail of near infra-red fluorophore-antibody conjugates targeted against key molecular (surface) markers of different cells in the TME, we are able to capture real-time TME dynamics at cellular resolution in two pre-clinical models; 1) a xenograft orthotopic mouse model of peritoneal carcinomatosis (disseminated metastases within the abdominal cavity) and imaging epidermal growth factor receptor (EGFR), CD44, CA125 (MUC16), transferrin receptor, Thomsen-Friedenreich carbohydrate antigen (T antigen), and CD45, 2) A syngeneic immunocompetent KPC cell line implanted mouse model of pancreatic ductal adenocarcinoma and imaging CD3, CD4, CD8a and CD45. Imaging on these tumor models was performed pre- and post-sub-therapeutic verteporfin (benzoporphyrin derivative monoacid A) photodynamic therapy (PDT, a cytotoxic light-based therapy). PDT treatment resulted in reduction of cancer cell burden, immune cell infiltration and alterations in their relative spatial localization. The results were confirmed by histopathological validation and ex vivo immunofluorescence staining of tumor tissue sections. Collectively, these results demonstrate the capability of HFME to image cancer cell phenotypes and the tumor microenvironment, in real time in live mice. With the ability to monitor cancer growth and treatment effects at a cellular level, HFME can potentially assist in customizing therapies in a patient-specific manner. Citation Format: Mohammad A. Saad, Bryan Q. Spring, Akilan Palanisami, Eric M. Kercher, Ryan T. Lang, Jason Sutin, Zhiming Mai, Tayyaba Hasan. Real-time visualization of tumor cell phenotype and microenvironmental heterogeneity enabled by a hyperspectral fluorescence microendoscope [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P044.

Hypoxia Drives Centrosome Amplification in Cancer Cells via HIF1α-dependent Induction of Polo-Like Kinase 4.

Hypoxia drives CA in cancer cells by regulating expression of PLK4, uncovering a novel HIF1α/PLK4 axis.

PD-1H Expression Associated With CD68 Macrophage Marker Confers an Immune-Activated Microenvironment and Favorable Overall Survival in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma (EC) in China. Although the PD-1 inhibitor pembrolizumab has been approved to treat patients with EC, its therapeutic efficacy is limited. Thus, additional immunotherapeutic targets for EC treatment are needed. Programmed Death-1 Homolog (PD-1H) is a negative checkpoint regulator that inhibits antitumor immune responses. Here, PD-1H expression in 114 patients with ESCC was evaluated by immunohistochemistry. Next, 12 randomly selected tumor tissue sections were used to assess the colocalization of PD-1H protein and multiple immune markers by multiplex immunohistochemistry. Our results demonstrated that PD-1H was expressed at high frequency in ESCC tumor tissues (85.1%). PD-1H protein was predominantly expressed in CD68+ tumor-associated macrophages and expressed at low levels in CD4+ T cells and CD8+ T cells in ESCC tumor tissues. Furthermore, based on ESCC data in The Cancer Genome Atlas (TCGA), the gene expression levels of PD-1H were positively associated with the infiltration levels of immune-activated cells especially CD8+ cytotoxic T cells. In contrast, the gene expression levels of PD-1H were negatively correlated with myeloid-derived suppressor cells (MDSCs). Importantly, PD-1H expression in tumor sites was significantly correlated with favorable overall survival in patients with ESCC. Collectively, our findings first provided direct information on the PD-1H expression pattern and distribution in ESCC, and positive correlation of PD-1H expression with overall survival suggested PD-1H expression levels could be a significant prognostic indicator for patients with ESCC. Future studies need to explore the immunoregulatory of PD-1H in the tumor microenvironment of ESCC.

TRPV2 Promotes Cell Migration and Invasion in Gastric Cancer via the Transforming Growth Factor-β Signaling Pathway.

Transient receptor potential vanilloid 2 (TRPV2) is a highly Ca2+-permeable ion channel that is involved in a number of cellular processes. It is expressed in various human cancers; however, the role of TRPV2 in gastric cancer (GC) remains poorly understood. TRPV2 gene expression was knocked down in GC cell lines by small-interfering RNA (siRNA), and the biological roles of TRPV2 in the proliferation, migration, and invasion of GC cells were then investigated. The gene expression profile of GC was elucidated using a microarray analysis. TRPV2 expression in tumor tissue sections was analyzed by immunohistochemistry. The migration and invasion abilities of GC cells were inhibited by the knockdown of TRPV2. Moreover, the microarray assay revealed that TRPV2 was associated with the transforming growth factor (TGF)-β signaling pathway. Immunohistochemical staining showed that the strong expression of TRPV2 correlated with lymphatic invasion, venous invasion, pathological T (pT), pathological N (pN), and a poor prognosis in GC patients. TRPV2 appeared to promote tumor migration and invasion via the TGF-β signaling pathway, and the strong expression of TRPV2 was associated with a worse prognosis in GC patients.

An aggregation-induced emission fluorescence probe for evaluating the effect of CYP450 changes under tumor chemotherapy

Cancer is a complex disease with very high incidence and mortality rates every year. However, cancer drug resistance greatly mitigates the cure rates of tumors, and cytochrome P450 (CYP450) plays an important role in the development of cisplatin resistance. We developed the aggregation-induced emission luminogen (AIEgen) TPE-CYP to monitor the changes in CYP450. The TPE-CYP fluorescent probe was successfully used to assess CYP450 levels in tumor cells and tumor tissue sections. This study presented that CYP450 level in HepG2/DDP cells (cisplatin-resistant cells) was higher than that in HepG2 cells, and the inhibition of CYP450 by 1-ABT effectively improved the tumor resistance. Thus, CYP450 plays a key role in the development of tumor resistance. The synergistic effect of 1-ABT and the chemotherapeutic agent cisplatin was superior to that of cisplatin alone in tumor-bearing mice. The TPE-CYP probe will provide an idea for the clinical implementation of individualized tumor treatment strategies, through the accurate monitoring of CYP450.

Pacific: A Phase II Study of Brentuximab Vedotin and Nivolumab Alone and then Combined with Rituximab, Cyclophosphamide, Doxorubicin and Prednisone for Patients with Untreated Primary Mediastinal Large B-Cell Lymphoma

Pacific: A Phase II Study of Brentuximab Vedotin and Nivolumab Alone and then Combined with Rituximab, Cyclophosphamide, Doxorubicin and Prednisone for Patients with Untreated Primary Mediastinal Large B-Cell Lymphoma

309 Visualizing the immunotherapy-induced spatial reorganization of the tumor-immune microenvironment by CODEX multiplex imaging

BackgroundTumors contain spatially organized microenvironments in which the location and composition of the cellular components ultimately determine tumor fate. We previously characterized CD45+ cells infiltrating T3 sarcomas using complementary forms of high-dimensional profiling (scRNAseq and CyTOF) and identified key immune cell populations that became associated either with growing tumors in mice treated with control antibody (cmAb) or rejecting tumors in mice treated with immune-checkpoint therapy (ICT) i.e., anti-PD-1 and/or anti-CTLA-4. To better understand the effects of the intratumoral immune cell populations on one another and on the tumor itself, we used CODEX multiplex imaging to simultaneously characterize expression of 35 distinct immune cell markers on tumor tissue sections thereby maintaining the spatial relationships between effector cells and their cognate tumor cell targetsMethodsT3 tumor bearing syngeneic mice were treated with cmAb or a-PD-1/a-CTLA-4 on days 6,9 and 12, harvested on different days, fresh frozen, cut in 8µM sections, transferred to coverslips, and stained with a panel of commercially available and custom-made CODEX antibodies following optimized staining conditions using the manufacturer’s protocol. Whole tumor tissue raw TIFF images were subjected to an image processing pipeline developed by Akoya Biosciences, Inc, where dapi+ cells used for cell segmentation by the Startdist method. Unsupervised clustering using Scanpy toolkit based on normalized marker expression profiles identified and validated 12 unique cell clustersResultsWhen compared to the cmAb group, a-PD-1/a-CTLA-4 tumors harvested on day 10 displayed a marked increase in percentage as well as density of CD4+ and CD8+ T cells, Ly6G neutrophils, MHC-II+cd11c+cd24+DCs and a decrease of CD140a+ tumor cells. Spatial analysis indicated statistically significant differences in the organization of these cell types upon treatment with a-PD-1/a-CTLA-4 compared to cmAb treatment. Specifically, immune cells were found at the border of the tumor in cmAb treated mice and heavily infiltrated tumors following ICT. Neighborhood identification analysis revealed changes in the composition of the main neighborhood clusters, of which the top three clusters present in cmAb treated tumors (macrophage, tumor, and other immune cells) were substituted by clusters of CD4+ and CD8+ T cells, neutrophils, and macrophages.ConclusionsThese results not only confirm the remodeling of both the lymphoid and myeloid compartments previously observed with scRNAseq and CyTOF but also suggest that changes in the spatial organization of immune cells are drivers of the tumor rejection process upon treatment with ICT.