Tumor Pieces Research Articles

Raman fiber-optic probe for rapid diagnosis of gastric and esophageal tumors with machine learning analysis or similarity assessments: a comparative study.

Gastric and esophageal cancers, the predominant forms of upper gastrointestinal malignancies, contribute significantly to global cancer mortality. Routine detection methods, including medical imaging, endoscopic examination, and pathological biopsy, often suffer from drawbacks such as low sensitivity and laborious and complex procedures. Raman spectroscopy is a non-invasive and label-free optical technique that provides highly sensitive biomolecular information to facilitate effective tumor identification. In this work, we report the use of fiber-optic Raman spectroscopy for the accurate and rapid diagnosis of gastric and esophageal cancers. Using a database of 14,000 spectra from 140 ex vivo tissue pieces of both tumor and normal tissue samples, we compare the random forest (RF) and our established Euclidean distance Raman spectroscopy (EDRS) model. The RF analysis achieves a sensitivity of 85.23% and an accuracy of 83.05% in diagnosing gastric tumors. The EDRS algorithm with improved diagnostic transparency further increases the sensitivity to 92.86% and accuracy to 89.29%. When these diagnostic protocols are extended to esophageal tumors, the RF and EDRS models achieve accuracies of 71.27% and 93.18%, respectively. Finally, we demonstrate that fewer than 20 spectra are sufficient to achieve good Raman diagnostic accuracy for both tumor tissues. This optimizes the balance between acquisition time and diagnostic performance. Our work, although conducted on ex vivo tissue models, offers valuable insights for in vivo in situ endoscopic Raman diagnosis of gastric and esophageal cancer lesions in the future. Our study provides a robust, rapid, and convenient method as a new paradigm in in vivo endoscopic medical diagnostics that integrates spectroscopic techniques and aRaman probe for detecting upper gastrointestinal malignancies.

Effects of IFN-γ on the immunological microenvironment and TAM polarity in stage IA non-small cell lung cancer and its mechanisms

ObjectiveTo investigate the effect of interferon-γ (IFN-γ) on the immune microenvironment and the polarity of tumor-associated macrophages (TAMs) in stage IA non-small cell lung cancer (NSCLC) and its mechanisms.MethodsHuman non-small cell lung cancer A549 cells were treated with a series of IFN-γ concentrations (0, 50, 100, 150, 200, 250, and 300 ng/mL). Tumor tissues from patients with stage IA NSCLC were cultured using the air–liquid interface culture technique to establish a tumor microenvironment (TME) organ model. The NSCLC model was constructed by subcutaneously embedding small tumor pieces into the back of nonobese diabetic severe combined immune deficiency (NOD SCID) mice. The size and weight of the tumors were recorded, and the tumor volume was calculated. CCK-8 assays were used to investigate cell proliferation, flow cytometry and TUNEL staining were used to evaluate cell apoptosis, colony formation was investigated by cloning experiments, and cell invasion and migration were evaluated by Transwell assays and scratch tests. The expression of apoptosis-related proteins (Bax, Bcl-2 and C-caspase 3), M2 polarization-related markers (CD163, CD206 and IDO1), and marker proteins of cytotoxic T cells and helper T cells (CD8 and CD4) was detected by Western blot. The expression of Ki-67 and IDO1 was detected by immunohistochemistry, and the levels of IL-6, IL-10, IL-13 and TNF-α were measured by ELISA. The expression of CD68 was measured by RT‒qPCR, and the phagocytosis of TAMs was evaluated by a Cell Trace CFSE kit and cell probe staining.ResultsThe proliferation activity of A549 cells increased with increasing IFN-γ concentration and peaked when the concentration reached 200 ng/mL, and the proliferation activity of A549 cells was suppressed thereafter. After treatment with 200 ng/mL IFN-γ, the apoptosis rate of cells decreased, the number of cell colonies increased, the invasion and migration of cells were promoted, the expression of Bax and C-caspase 3 was downregulated, and the expression of Bcl-2 was upregulated in cells and the TME model. In the TME model, CD163, CD206, IDO1 and Ki-67 were upregulated, CD8 and CD4 were downregulated, apoptosis was reduced, the levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased. IL-4 induced TAMs to express CD163 and CD206, reduced the levels of IL-6 and TNF-α, increased the levels of IL-10 and IL-13, and weakened the phagocytic function of TAMs. IFN-γ treatment further enhanced the effect of IL-4 and enhanced the viability of A549 cells. IDO1 decreased the viability of T cells and NK cells, while suppressing the effect of IFN-γ. In mice, compared with NSCLC mice, the tumor volume and weight of the IFN-γ group were increased, the expression of CD163, CD206, IDO1, Ki-67 and Bcl-2 in tumor tissue was upregulated, the expression of Bax and C-caspase 3 was downregulated, and apoptosis was reduced. The levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased in the serum of mice.ConclusionIn stage IA NSCLC, a low concentration of IFN-γ promotes the polarization of TAMs to the M2 phenotype in the TME model by upregulating the expression of IDO1, promoting the viability of cancer cells, inhibiting the viability of T cells and NK cells, and thus establishing an immune microenvironment conducive to tumor progression.

P12.14.B HIGHLY INVASIVE GLIOMA CELLS MOTILITY MEASUREMENTS ON A VARIETY OF EXTRACELLULAR MATRIX PROTEINS

Abstract BACKGROUND Glioblastoma multiforme (GBM) is a heterogeneous highly invasive malignant brain tumor. The extracellular matrix (ECM) produced by tumor microenvironment plays an important role in the regulation of cell motility and invasion into surrounding healthy tissue. In this research we determined the effect of various ECM proteins on the motility of primary tumor cells. MATERIAL AND METHODS Primary tumor cells were isolated from biopsy material obtained during GBM surgical removal. Cultivation of the piece of tumor (0.5 mm3) was carried out in the form of an 3D explant model at 37°C in 5% CO2-incubator. After several months, cells migrated from the tumor spontaneously formed spheroids. To assess the rate of cell migration, spheroids were seeded on 24-well plate coated with poly-L-lysine, fibrinogen, collagen type I, and Matrigel. After 3 days, the field of view in the well (n=9) was microphotographed every 15 min for 56 hours, after which the tracks of cells outside the spheroids were manually marked (n=50) on the obtained images. The average and maximum speeds were calculated, as well as the straightness of the track (the ratio of the Euclidean distance to the track length). The Mean square displacement (MSD) was plotted against time for each protein studied in comparison with random walk simulation calculated for the equal track parameters (number of steps, mean and sd) on correspondent protein. RESULTS Live-cell imaging of the patient-derived cells revealed the heterogeneous population of actively moving cells outside the spheroids. The following results of measuring cell motility were obtained (mean ± sd): (1) Mean speed on poly-L-lysine 4.3 ± 2.5 μm/h; on fibrinogen 11.1 ± 5.7 μm/h; on collagen type I 8.8 ± 5.1 μm/h; on Matrigel 9.7 ± 3.6 μm/h. (2) Max speed on poly-L-lysine 63.8 ± 30.1 μm/h; on fibrinogen 90.0 ± 38.9 μm/h; on collagen type I 96.1 ± 42.1 μm/h; on Matrigel 84.1 ± 34.1 μm/h. (3) Straightness on poly-L-lysine 0.24 ± 0.14 μm/h; on fibrinogen 0.18 ± 0.11 μm/h; on collagen type I 0.14 ± 0.09 μm/h; on Matrigel 0.20 ± 0.12 μm/h. MSD analysis showed the greatest contribution of random walk in the motility of cells on collagen type I, the smallest - on Matrigel. CONCLUSION Proteins of ECM affect not only the speed of cell movement, but also the tortuosity of their tracks. Further studies will clarify the mechanisms of GBM invasion depending on the composition of the ECM.

Abstract 3100: XenoSarc: patient-derived xenograft (PDX) models of soft tissue sarcoma (STS) and their histopathological and molecular characterization

Abstract STS constitutes a family of rare mesenchymal tumors with more than 70 subtypes described. The limited treatment options available for advanced STS patients underline the need for reliable preclinical models to test new therapeutic approaches. We established a panel of PDX models (XenoSarc) by subcutaneous implantation of fresh tumor specimens in athymic mice (nu/nu NRMI). Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of animals. At each passage tumor fragments were collected for histopathological and molecular characterization. In an ongoing effort 493 STS samples from 414 consenting patients treated at the University Hospitals, Leuven (Belgium) have been transplanted. A total of 67 PDX models from 20 STS subtypes have been established, meaning they have stable morphological, immunohistochemical and genetic characteristics over at least 2 passages. The PDX platform includes more common STS subtypes such as myxofibrosarcoma (n=12 models), gastrointestinal stromal tumors (9), dedifferentiated liposarcoma (10), and leiomyosarcoma (8), as well as models from ultra-rare subtypes, e.g. pulmonary intimal sarcoma, extraskeletal osteosarcoma, mesenchymal chondrosarcoma, myxoinflammatory fibroblastic sarcoma and others. All relevant details about the donor patient and tumor characteristics, including sensitivity to the standard treatments, are known for every model. The models are well-characterized, with availability of molecular information on genomic profile (by low-coverage whole genome sequencing), and expression profile (by RNA sequencing). Xenografts are accompanied by ready to use tissue microarrays (TMA) from the models, which can be exploited for target identification and model selection for preclinical studies. Ex-mouse material can also be used to establish primary cell cultures and 3D organoids for in vitro screening purposes. The XenoSarc platform offers opportunities for studying the biology of various sarcoma subtypes including ultra-rare entities and was found to be a very reliable tool for early drug screening in STS in preparation of clinical testing of novel compounds. The platform is available for collaborative preclinical projects with academic and industrial partners. Citation Format: Agnieszka Wozniak, Luna De Sutter, Lore De Cock, Britt Van Renterghem, Che-Jui Lee, Yannick Wang, Ulla Vanleeuw, Kimberly Verbeeck, Daphne Hompes, Friedl Sinnaeve, Hazem Wafa, Baki Topal, Joris Jaekers, Dirk Van Raemdonck, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. XenoSarc: patient-derived xenograft (PDX) models of soft tissue sarcoma (STS) and their histopathological and molecular characterization [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 3100.

Abstract 1754: Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models

Abstract Background: Amatoxin-based antibody-drug conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the present study, we show that ATACs belong to the class of immune activating drugs which exhibit synergistic anti-tumor efficacy with immune checkpoint inhibitors (ICIs) in vivo by induction of immunogenic cell death (ICD). ICIs are a new class of cancer therapeutics utilizing patients` immune system to kill cancer cells. ICIs rely on the activity of the immune system to develop their full potential. Therefore, drugs that are heating up cold tumors and make them visible to the patients` immune system and by that enhancing the anti-tumor efficacy of ICIs, e.g., ATACs, are on high demand for the treatment of tumor patients. Material and methods: Cell line: Her2+ cell lines: NCI-N87; CD19+ cell line: Raji Antibody: anti-Her2 and anti-CD19 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Animal models: Raji cells with human peripheral blood mononuclear cells (PBMCs), NCI-N87 cells or HCBx-10/HCBx-11 PDX (Xentech, France) tumor pieces were implanted subcutaneously in mice. Treatment: ATAC: single dose i.v.; immune checkpoint inhibitors: Q3Dx6 or Q3Dx5. Results: In vivo treatment of heterogenous Her2low PDX tumors with an anti-Her2-ATAC led to a significant tumor growth delay and complete tumor remission. Furthermore, increased expression of HMGB1 and surface exposure of calreticulin (CRT), two hallmarks of ICD, was observed in the same PDX tumors as well as in a Her2+ CDX (NCI-N87) tumor after the treatment with an anti-HER2 ATAC. In addition, the combined treatment of ATACs and different ICIs targeting CTLA-4 (Ipilimumab), PD-L1 (Pembrolizumab) or PD-1 (Avelumab) had a synergistic effect in a humanized lymphoma CDX model in the presence of human PBMCs. Combination treatment led to enhanced tumor growth inhibition and more tumor free animals as compared to single treatments with ATAC or ICI. Conclusions: The strong anti-tumor efficacy of ATACs even in heterogenous patient-derived xenograft models in combination with the induction of ICD in vitro and in vivo suggest that the anti-tumor effect of ATACs is accompanied by the activation of the immune system. This hypothesis is strengthened by the finding that ATACs and ICIs show a synergistic effect in vivo. The presented data highlights the general concept of the synergistic effect of ATACs and ICIs which applies to several types of ICIs thus strengthening the scientific rationale for combination treatments in clinical trials. Citation Format: Christian Orlik, Franziska Ebeling, Kristin Decker, Irina Dranova, Anikó Pálfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models [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 1754.

Abstract P5-02-03: Phenotypic analysis for TNBC using a novel breast cancer microphysiological system

Abstract Breast cancer (BC) is among the most commonly diagnosed cancers in women and is the leading cause of malignant death in U.S. women. The triple negative breast cancer (TNBC) subtype is more aggressive and has a poorer prognosis compared to other subtypes. Patient derived xenografts (PDX), human tumors transplanted and grown in mice, are a newer, better model of TNBC. However, there are barriers when using mouse models: mice stroma can take over the PDX tumors, using immunocompromised mice can prevent immune responses and site-specific interaction, and mice are expensive. To overcome these barriers, a translational microphysiological system (MPS) was developed that is capable of maintaining the primary, human breast microenvironment in vitro. By seeding these MPS with breast cancer cell lines or tumor explants, we produce breast cancer MPS (BC-MPS). Here, we show the model’s capabilities of supporting PDX tumor viability through flow cytometry on multiple PDX models as well as effects of human breast tissue on TNBC cell lines viability and proliferation. After showing the BC-MPS’ stability ex vivo, the effects on the cancer stem cell (CSC) population and lipid accumulation were explored through flow cytometry. Tu-BcX-4IC was seeded alone and in BC-MPS. After 7 days of incubation, the tumor was removed and the CSC population and lipid accumulation was measured through flow cytometry, showing an increase in CSC population and lipid accumulation when the PDX tumor was cultured in BC-MPS compared to cultured in 2D. Architectural changes that occur with the PDX explants when placed in BC-MPS were also monitored. The TNBC PDX model, Tu-BcX-4IC, was excised and seeded alone or in the BC-MPS system. The human breast tissue (HBT) and a PDX tumor piece was initially fixed in formalin before placed into BC-MPS (Day 0). After 4, 7, and 14 days of incubation, the 4IC PDX tumor explants seeded alone and in BC-MPS were fixed in formalin, paraffin embedded, and sectioned for H&E staining to determine the effect of the surrounding HBT on the PDX tumor. In additions to demonstrating the model’s capabilities of supporting PDX tumor viability and examining tumor architectural changes, assays were performed to test experimental capabilities of BC-MPS. Different BC cell lines were placed into the system and treated with chemotherapeutics to determine if the microphysiological system is capable of pharmaceutical studies. Here, we focus on the TNBC cell lines which were seeded alone and in the BC-MPS, and their response to the chemotherapeutic drugs, Paclitaxel, Romidepsin, and Cobimetinib. The proliferation response was monitored by luciferase imaging, demonstrating that BC-MPS can be used for drug studies.BC-MPS is a promising new translational microphysiological system that facilitates studying long term interactions between real human breast tissue and cancer cells as well as the native tumor environment in HBT. The BC-MPS system’s ability to support the growth of established cell lines as well as PDXs has been demonstrated. Future studies will focus on showing human tumor viability in BC-MPS, developing the model for personalized medicine. Citation Format: Katherine Hebert, Khoa Nguyen, Thomas Cheng, Madlin Alzoubi, Bridgette Collins-Burow, Elizabeth Martin, Frank Lau, Matthew Burow. Phenotypic analysis for TNBC using a novel breast cancer microphysiological system [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 P5-02-03.

Fluorescence-guided craniotomy of glioblastoma using panitumumab-IRDye800.

A contrast-enhancing lesion in the left temporal lobe of a 72-year-old woman was biopsied and diagnosed as glioblastoma. Near-infrared (NIR)–labeled epidermal growth factor receptor (EGFR) antibody, panitumumab-IRDye800, was infused 52 hours before craniotomy without pretreatment. Tumor fluorescence was detected through intact dura, and the visual contrast between disease and peritumoral healthy brain was enhanced after tumor exposure. Residual cancerous tissue was identified with strong fluorescence in resection cavity after en bloc tumor removal. Minimal fluorescence remained in the final wound bed, likely from nonenhancing tumor. Fluorescence was heterogeneously distributed at the infiltrative margin in resected tumor pieces imaged ex vivo. Postoperative MRI confirmed gross-total resection.The video can be found here: https://stream.cadmore.media/r10.3171/2021.10.FOCVID21201

The HROC-Xenobank-A High Quality Assured PDX Biobank of >100 Individual Colorectal Cancer Models.

Simple SummaryConsidering recent research, it was established that the best experimental models to conserve biological features of human tumors and to predict individual clinical treatment success are patient-derived xenografts (PDX). Their recognized and growing importance for translational research, especially for late-stage preclinical testing of novel therapeutics, necessitates a high number of well-defined PDX models from individual patients’ tumors. The starting platform for the Hansestadt Rostock colorectal cancer (HROC)-Xenobank was the assortment of colorectal tumor and normal tissue samples from patients stored in our university biobank.Based on our research group’s large biobank of colorectal cancers (CRC), we here describe the ongoing activity of establishing a high quality assured PDX biobank for more than 100 individual CRC cases. This includes sufficient numbers of vitally frozen (n > 30 aliquots) and snap frozen (n > 5) backups, “ready to use”. Additionally, PDX tumor pieces were paraffin embedded. At the current time, we have completed 125 cases. This resource allows histopathological examinations, molecular characterizations, and gene expression analysis. Due to its size, different issues of interest can be addressed. Most importantly, the application of low-passage, cryopreserved, and well-characterized PDX for in vivo studies guarantees the reliability of results due to the largely preserved tumor microenvironment. All cases described were molecularly subtyped and genetic identity, in comparison to the original tumor tissue, was confirmed by fingerprint analysis. The latter excludes ambiguity errors between the PDX and the original patient tumor. A cancer hot spot mutation analysis was performed for n = 113 of the 125 cases entities. All relevant CRC molecular subtypes identified so far are represented in the Hansestadt Rostock CRC (HROC)-Xenobank. Notably, all models are available for cooperative research approaches.

Intra-Tumour Genetic Heterogeneity and Prognosis in High-Risk Neuroblastoma.

Simple SummaryNeuroblastoma (NB) is the most common extra-cranial solid paediatric cancer and is responsible for 15% of childhood cancer deaths. Patients with NB are characterized by presenting a very heterogeneous clinic (inter-tumoural heterogeneity) and also both spatial and temporal intra-tumour heterogeneity (ITH) reflected in their genetic aberrations, which may be the consequence of the coexistence of different microenvironments within the tumour. Applying pangenomic techniques to detect genomic aberrations in different biopsies (solid and liquid) of high risk NB (HR-NB) we have detected spatial ITH in a surprisingly high percentage (almost 40%) of the studied cohort. Moreover, a positive association between this heterogeneity and survival has been found. Confirming these results, combining tumour material analysis in a large cohort of HR-NB will have a major impact in the genetic diagnosis routine procedure, and would also entail a revision of the prognosis of patients with ITH.Spatial ITH is defined by genomic and biological variations within a tumour acquired by tumour cell evolution under diverse microenvironments, and its role in NB patient prognosis is understudied. In this work, we applied pangenomic techniques to detect chromosomal aberrations in at least two different areas of each tumour and/or in simultaneously obtained solid and liquid biopsies, detecting ITH in the genomic profile of almost 40% of HR-NB. ITH was better detected when comparing one or more tumour pieces and liquid biopsy (50%) than between different tumour pieces (21%). Interestingly, we found that patients with ITH analysed by pangenomic techniques had a significantly better survival rate that those with non-heterogeneous tumours, especially in cases without MYCN amplification. Moreover, all patients in the studied cohort with high ITH (defined as 50% or more genomic aberration differences between areas of a tumour or simultaneously obtained samples) survived after 48 months. These results clearly support analysing at least two solid tumour areas (separately or mixed) and liquid samples to provide more accurate genomic diagnosis, prognosis and therapy options in HR-NB.

P70.04 Results From a Patient Avatar Program Utilizing Murine Xenografts and Organoids After Neoadjuvant Therapy for Operable NSCLC

Patient-derived xenografts (PDX) and organoids (PDO) are well described methods of propagating patient tumors. While propagation of such models are well established in treatment naïve patients after surgical resection, little is known of the prognostic utility of such methods in the context of patients who have undergone neoadjuvant therapy with conventional therapy (chemotherapy +/- radiation), immune checkpoint blockade (ICB) or tyrosine kinase inhibitor (TKI) for patients with operable non-small cell lung cancer (NSCLC). This study aims to evaluate PDX and PDO propagation rates after surgical resection from patients treated with a variety of conventional and emerging ICB neoadjuvant treatment regimens, as well as correlation with extent of pathological response. All trials of PDX and PDO implantation performed at the McGill University, Goodman Cancer Research Centre since 2017 from samples of lung cancer patients treated at the Montreal General Hospital were reviewed. After intraoperative specimen evaluation by a clinical pathologist, tumor pieces of 5 mm3 were taken for live banking PDX and PDO procedures. A retrospective chart review was then performed to obtain patient demographic and treatment data. A total of 80 PDX implantation and 28 PDO culture trials were performed, with respective take rates of 25% and 82%. Overall, 35/80 PDX trials (20 chemotherapy only, 2 chemotherapy +/- ICB, 2 chemoradiation, 10 ICB, and 1 TKI) and 23/28 PDO trials (11 chemotherapy only, 3 chemotherapy +/- ICB, 1 chemoradiation, 7 ICB, and 1 TKI) were from patients who received neoadjuvant treatment. In these patients, our take rates were 17% for PDX and 78% for PDO. 5 neoadjuvant-treated patients had a major pathological response (MPR) and 2 patients, a pathological complete response (pCR). Of those with MPR, 1/5 sample was successfully propagated in PDXs, and another 1/5 sample failed to propagate in PDXs, but grew organoids. Interestingly, 0/10 patients who received neoadjuvant ICB grew in PDXs versus 3/10 where PDOs were successfully propagated. PDX and PDO models offer tangible opportunities to create living patient avatars. Our study suggests that PDX and PDO propagation is possible after a variety of neoadjuvant treatment regimens, though we were unable to propagate PDX from ICB-treated patients. Furthermore, MPR did not preclude subsequent tumor propagation in PDX or PDO. The clinical relevance of these findings and biology underlying our findings remains to be further explored.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non‐adhesive alginate. High‐throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal‐ and solid‐like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick‐wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks’ culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high‐throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.

Natural killer cells and cytotoxic T lymphocytes are required to clear solid tumor in a patient-derived xenograft.

Existing patient-derived xenograft (PDX) mouse models of solid tumors lack a fully tumor donor–matched, syngeneic, and functional immune system. We developed a model that overcomes these limitations by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in 83% to 89% of TIL-PDX mice, and these were seen to harbor exhausted immuno-effector as well as functional immunoregulatory cells persisting for at least 6 months postengraftment. Combined treatment with interleukin-15 stimulation and immune checkpoint inhibition resulted in complete or partial tumor response in this model. Further, depletion of cytotoxic T lymphocytes and/or natural killer cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.

Abstract 2996: Creating PDXs from continental African breast tumors: addressing racial inequity through science

Abstract To design interventions to reduce the burden of breast cancer (BC) in women with African ancestry, there is the need to understand the factors - heritable or non-heritable, modifiable or non-modifiable - that are associated with the disease. Patients and tumors from black women have been underrepresented in clinical research which are the main route to identifying new and improved treatments. Triple negative breast cancer (TNBC) is a clinically aggressive form of BC with propensity to metastasize and is enriched in patients with hereditary predisposition to BC. TNBC is most prevalent among black women despite lacking strong links with the known BC. This phenotype prevalence contributes to disparities in BC outcomes between blacks and other populations. There is an urgent need to represent black tumors in clinical research. Here we describe the process we used to transport fresh breast tumors from Ghana, west Africa to the USA to develop PDXs. Considering the shared ancestry between west Africans and diasporan African American, the results of this work will benefit both populations. Through an international collaboration between investigators at University of Michigan (UM) and Komfo Anokye Teaching Hospital (KATH) in Ghana, regular team site visits were initiated to generate surgical specimens for transport to the United States of America (USA). These specimens were used to create in vivo models. With informed consent, excess patient tumor tissue was taken from the operating room, at KATH. The tumors were cut into small pieces, and slow- frozen to -80°C in 10% DMSO/90%FBS. The samples were then transported overnight on dry ice in a commercial flight to UM. At UM, the tumor pieces were rapidly thawed and rinsed off with HBSS or PBS. The tumor sample was cut into smaller fragments (approximately 1-2 mm in diameter), mixed with matrigel (BD), and 25 μl of matrigel per injection site was injected into the mammary fat pad of female NOD/scid/IL2Rgnull (NSG) mice with an 18-gauge needle. From 2012 to 2017, we obtained fresh tumor samples from 49 Ghanaian patients. Of these, 23 (47%) were TNBC and 26 (53%) were other subtypes. Of the 23 TNBC samples, we implanted 18 into NSG mice and developed PDXs from 12. This gave us a success rate of 67%. We have fully characterized these tumors and their corresponding primary tumors. Our results demonstrate a maintenance of the histology and marker expression in the corresponding PDXs. Additionally, we obtained samples and developed PDXs from 17 Caucasian Americans, 13 African Americans, and 2 Asian patients from UM. 20 samples were TNBCs and 13 were non-TNBC. 18 were implanted in mice (12 TNBC, 4 ER+, and 2 Her2+). Of these, 11 developed into PDXs (9 TNBC, 2 ER+). Transporting African tumors to the USA is expensive, challenging, and limits large scale studies. These challenges can be overcome by enhancing African based research resources in order to conduct research in Africa. Citation Format: Evelyn M. Jiagge, Tahra K. Suhan, Jessica M. Bensenhaver, Michele Dziubinski, Joseph K. Oppong, Francis Aitpillah, Awuah Baffour, John D. Carpten, Lisa A. Newman, Sofia D. Merajver, Max S. Wicha. Creating PDXs from continental African breast tumors: addressing racial inequity through science [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2996.

Tension-Free Anterior Rerouting of the Facial Nerve in Management of Jugular Foramen Paragangliomas.

Tension-Free Anterior Rerouting of the Facial Nerve in Management of Jugular Foramen Paragangliomas.

Creation and Maintenance of a Living Biobank - How We Do It.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

CircRNA circFAT1(e2) Elevates the Development of Non-Small-Cell Lung Cancer by Regulating miR-30e-5p and USP22.

Background As a newly discovered regulatory RNA, circular RNA (circRNA) has become a hot spot in many tumor pieces of research. In recent years, it has been discovered that circRNAs have multiple biological effects in different stages of cancer. However, the expression pattern and mechanism of circFAT1(e2) in non-small-cell lung cancer (NSCLC) are still unclear. Methods The expressions of circFAT1(e2) in NSCLC tissues and cell lines were studied. Functionally, CCK-8 and transwell experiments were performed in A549 and H1299. In addition, we also performed a dual-luciferase report analysis to clarify the mechanism of action of circFAT1(e2). Results circFAT1(e2) was significantly upregulated in NSCLC tissues and cell lines. circFAT1(e2) gene knockdown could significantly inhibit the proliferation, migration, and invasion of NSCLC cells. Loss of function testing found that circFAT1(e2) functioned as an oncogene in NSCLC cells. In addition, circFAT1(e2) acted as a ceRNA to spongy miR-30e-5p, which led to the increase in USP22 and promoted cell growth. Conclusions The circFAT1(e2)-miR-30e-5p-USP22 axis is a crucial part of the progression of NSCLC. This study suggests that circFAT1(e2) may be an important potential of prognostic prediction and treatment targets for NSCLC patients.

3D Collagen Vascular Tumor-on-a-Chip Mimetics for Dynamic Combinatorial Drug Screening.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

Non image-guided core needle biopsies can be used safely to improve diagnostic efficiency for soft tissue tumors

IntroductionFront-line biopsy remains the rule in the management of soft tissue mass syndromes. Although open biopsy has long been considered the gold standard, it has recently been shown that a percutaneous biopsy is associated with a reduction in the rate of complications and cost, while maintaining high diagnostic accuracy. Though there is much literature regarding the diagnostic accuracy of image-guided and open biopsies for soft tissue tumors, the accuracy of percutaneous non image-guided biopsies has not been well documented. The objective of this study was to compare the failure rate of non image-guided biopsies, image-guided biopsies and open biopsies for the diagnosis of soft tissue tumors. We also attempted to identify the failure risk factors for non image-guided biopsies and we compared the diagnostic delay of the three types of biopsy. Materials and methodsThis was a continuous, single-center retrospective study. We reviewed the results from 337 patients managed with a biopsy (percutaneous or open) for a soft tissue tumor, all carried out in our center between January 2010 and December 2015. Biopsy technique was chosen by the treating orthopedic surgeon, according to the clinical and radiological characteristics of the mass. 141 patients (41.8%) had a non-image-guided biopsy as the first-line diagnostic procedure, 81 (24.0%) had an image-guided biopsy, and 115 (34.1%) an open biopsy. Diagnostic failure was defined either by a non-contributory biopsy, the need for repeat biopsy, or a major histological discordance obtained from the resected tumor piece. The risk factors studied were tumor characteristics, patient’ characteristics and sampling modalities. Diagnostic delay was defined as the period between the day of the first external consultation at the hospital and the day of the notification of the diagnosis by the physician. ResultsWe obtained a failure rate of 9.9% (14 patients) for non image-guided biopsies. Eleven were non-contributive and three were considered as errors of diagnosis. The failure rate for image-guided biopsies was 18.5% (15 patients), with no significant difference compared with non image-guided biopsies. The open biopsies were associated with a failure rate of 6.9% (eight patients). We found no failure risk factors for non image-guided biopsies. Diagnostic delay was significantly shorter for non image-guided biopsies (p = 0.001). ConclusionWhen performed in a referral center by the patient's surgeon, a non-image-guided core needle biopsy is a safe procedure which ensures equivalent diagnostic accuracy for soft tissue tumors, while reducing the diagnostic delay.

Disseminated Peritoneal Leiomyomatosis with Uterine Smooth Muscle Tumors of Uncertain Malignant Potential After Morcellator

Disseminated peritoneal leiomyomatosis (DPL) are characterized by the dissemination of smooth muscle nodules throughout the peritoneal surfaces. Smooth uterine muscle tumors of uncertain malignant potential (STUMP) is also a rare situation with complicated diagnosis and treatment with similar disease presentation to DPL. Interventional procedure such as laparoscopic morcellation can disseminate small tumor pieces all around the abdominal cavity. The objective of this study is to evaluate whether imminent follow-ups are needed after laparoscopic surgeries such as morcellation to reduce disease recurrence. We discuss the case of a 43-years-old patient presented with abdominal pain who had laparoscopic myomectomy 2 years ago. A uterine mass was noted with ultrasonography and computed tomography. The pathological results of masses was reported as STUMP. For both DPL and STUMP, diagnosis and treatments are difficult and problematic. This emphasizes the importance of following up with patients after laparoscopic surgeries, especially after morcellation.

SURG-24. NOVEL MALLEABLE FORCIPES FOR ENDOSCOPIC ASSISTED TECHNIQUE IN PEDIATRIC BRAIN TUMORS

Recent advances in optical devices and surgical instruments have been applied to neurosurgery. Even with modifications, one of the most serious risks is injury of neuronal and vascular structure caused by operation of surgical instruments in a narrow surgical field. Fixed instruments are not practical for pediatric brain tumor surgeries because the length of the curved or angled tip portion is limited because of the narrow entrance. We developed a novel malleable forceps to resolve the difficulties related to microsurgical procedures. The malleable forceps has two shafts with a sharp cup at the tip. The entire forceps was made of stainless steel, with a silver and nickel alloy inserted between 10 and 40 mm from the tip. In the alloy part, the surgeon can flex the forceps freely using a special cylinder. The special cylinder is useful to prevent from slipping of the cups of tip. The maximum angle that can be bent is 70 degrees vertically. We also developed a monoshaft malleable forceps. We used these flexible forcipes in the case of various pediatric brain tumors including craniopharyngioma. We performed tumor resection by anterior interhemispheric trans-lamina terminalis approach. After procedure of tumor resection using microscope, endoscope inserted around the pituitary stalk. The piece of calcified tumor could be easily removed without any complications. These forcipes can be deformed to an appropriate angle and can be applied to various cases, especially pediatric brain tumors.