Cancer Tissue-originated Spheroid Research Articles

Advancements in the Understanding of Small-Cell Neuroendocrine Cervical Cancer: Where We Stand and What Lies Ahead.

Small-cell neuroendocrine cervical carcinoma (SCNCC) is a rare yet aggressive gynecological malignancy associated with dismal clinical outcomes. Its rarity has led to a limited number of retrospective studies and an absence of prospective research, posing significant challenges for evidence-based treatment approaches. As a result, most gynecologic oncology centers have limited experience with this tumor, emphasizing the urgent need for a comprehensive review and summary. This article systematically reviews the pathogenesis, immunohistochemical and molecular characteristics, prognostic factors, and clinical management of gynecologic SCNCC. We specifically focused on reviewing the distinct genomic characteristics of SCNCC identified via next-generation sequencing technologies, including loss of heterozygosity (LOH), somatic mutations, structural variations (SVs), and microRNA alterations. The identification of these actionable genomic events offers promise for discovering new molecular targets for drug development and enhancing therapeutic outcomes. Additionally, we delve deeper into key clinical challenges, such as determining the optimal treatment modality between chemoradiation and surgery for International Federation of Gynecology and Obstetrics (FIGO) stage I phase patients within a precision stratification framework, as well as the role of targeted therapy within the homologous recombination (HR) pathway, immune checkpoint inhibitors (ICIs), and prophylactic cranial irradiation (PCI) in the management of SCNCC. Finally, we anticipate the utilization of multiple SCNCC models, including cancer tissue-originated spheroid (CTOS) lines and patient-derived xenografts (PDXs), to decipher driver events and develop individualized therapeutic strategies for clinical application.

Oxygen metabolism analysis of a single organoid for non-invasive discrimination of cancer subpopulations with different growth capabilities.

Heterogeneous nature is a pivotal aspect of cancer, rendering treatment problematic and frequently resulting in recurrence. Therefore, advanced techniques for identifying subpopulations of a tumour in an intact state are essential to develop novel screening platforms that can reveal differences in treatment response among subpopulations. Herein, we conducted a non-invasive analysis of oxygen metabolism on multiple subpopulations of patient-derived organoids, examining its potential utility for non-destructive identification of subpopulations. We utilised scanning electrochemical microscopy (SECM) for non-invasive analysis of oxygen metabolism. As models of tumours with heterogeneous subpopulations, we used patient-derived cancer organoids with a distinct growth potential established using the cancer tissue-originated spheroid methodology. Scanning electrochemical microscopy measurements enabled the analysis of the oxygen consumption rate (OCR) for individual organoids as small as 100µm in diameter and could detect the heterogeneity amongst studied subpopulations, which was not observed in conventional colorectal cancer cell lines. Furthermore, our oxygen metabolism analysis of pre-isolated subpopulations with a slow growth potential revealed that oxygen consumption rate may reflect differences in the growth rate of organoids. Although the proposed technique currently lacks single-cell level sensitivity, the variability of oxygen metabolism across tumour subpopulations is expected to serve as an important indicator for the discrimination of tumour subpopulations and construction of novel drug screening platforms in the future.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer

BackgroundPrecision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism.MethodsWe generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient’s cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug’s efficacies against HER2 E401G.ResultsWe confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective.ConclusionsAfatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Ex vivo chemosensitivity assay using primary ovarian cancer organoids for predicting clinical response and screening effective drugs.

Selecting the best treatment for individual patients with cancer has attracted attention for improving clinical outcomes. Recent progress in organoid culture may lead to the development of personalized medicine. Unlike molecular-targeting drugs, there are no predictive methods for patient response to standard chemotherapies for ovarian cancer. We prepared organoids using the cancer tissue-originated spheroid (CTOS) method from 61 patients with ovarian cancer with 100% success rate. Chemosensitivity assays for paclitaxel and carboplatin were performed with 84% success rate using the primary organoids from 50 patients who received the chemotherapy. A wide range of sensitivities was observed among organoids for both drugs. All four clinically resistant organoids were resistant to both drugs in 18 cases in which clinical response information was available. Five out of 18 cases (28%) were double-resistant, the response rate of which was compatible with the clinical remission rate. Carboplatin was significantly more sensitive in serous than in clear cell subtypes (P = 0.025). We generated two lines of organoids, screened 1135 drugs, and found several drugs with better combinatory effects with carboplatin than with paclitaxel. Some drugs, including afatinib, have shown an additive effect with carboplatin. The organoid sensitivity assay did not predict the clinical outcomes, both progression free and overall survival.

Antitumor effect of WEE1 blockade as monotherapy or in combination with cisplatin in urothelial cancer

Overcoming cisplatin (CDDP) resistance is a major issue in urothelial cancer (UC), in which CDDP‐based chemotherapy is the first‐line treatment. WEE1, a G2/M checkpoint kinase, confers chemoresistance in response to genotoxic agents. However, the efficacy of WEE1 blockade in UC has not been reported. MK‐1775, a WEE1 inhibitor also known as AZD‐1775, blocked proliferation of UC cell lines in a dose‐dependent manner irrespective of TP53 status. MK‐1775 synergized with CDDP to block proliferation, inducing apoptosis and mitotic catastrophe in TP53‐mutant UC cells but not in TP53‐WT cells. Knocking down TP53 in TP53‐WT cells induced synergism of MK‐1775 and CDDP. In UMUC3 cell xenografts and two patient‐derived xenograft lines with MDM2 overexpression, in which the p53/cell cycle pathway was inactivated, AZD‐1775 combined with CDDP suppressed tumor growth inducing both M‐phase entry and apoptosis, whereas AZD‐1775 alone was as effective as the combination in RT4 cell xenografts. Drug susceptibility assay using an ex vivo cancer tissue‐originated spheroid system showed correlations with the in vivo efficacy of AZD‐1775 alone or combined with CDDP. We determined the feasibility of the drug susceptibility assay using spheroids established from UC surgical specimens obtained by transurethral resection. In conclusion, WEE1 is a promising therapeutic target in the treatment of UC, and a highly specific small molecule inhibitor is currently in early phase clinical trials for cancer. Differential antitumor efficacy of WEE1 blockade alone or combined with CDDP could exist according to p53/cell cycle pathway activity, which might be predictable using an ex vivo 3D primary culture system.

Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma.

Micropapillary carcinoma (MPC) is a morphologically distinctive form of carcinoma, composed of small nests of cancer cells surrounded by lacunar spaces. Invasive MPC is associated with poor prognosis. The nests of tumor cells in MPC reportedly exhibit reverse polarity, although the molecular mechanisms underlying MPC patterns are poorly understood. Using the cancer tissue-originated spheroid (CTOS) method, we previously reported polarity switching in colorectal cancer (CRC). When cultured in suspension, the apical membrane promptly switches from the outside surface of the CTOSs to the surface of the lumen inside the CTOSs under extracellular matrix (ECM)-embedded conditions, and vice versa. Here, we investigated two CTOS lines from CRC patient tumors with MPC lesions. Xenograft tumors from the CTOSs exhibited the MPC phenotype. The MPC-CTOSs did not switch polarity in vitro. Time-course analysis of polarity switching using real-time imaging of the apical membrane revealed that local switching was continually propagated in non-MPC-CTOSs, while MPC-CTOSs were unable to complete the process. Integrin β4 translocated to the outer membrane when embedded in ECM in both MPC and non-MPC-CTOSs. Protein levels, as well as the active form of RhoA, were higher in MPC-CTOSs. The suppression of RhoA activity by GAP overexpression enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, while overexpression of active RhoA did not affect polarity switching in non-MPC-CTOSs. Pretreatment with a ROCK inhibitor enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, although delayed treatment after becoming embedded in ECM failed to do so. Thus, the inability to switch polarity might be a cause of MPC, in which the aberrant activation of RhoA plays a critical role. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Heterogenous chemosensitivity of a panel of organoid lines derived from small cell neuroendocrine carcinoma of the uterine cervix.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare disease with a poor prognosis. The lack of established disease models has hampered therapy development. We generated a panel of cancer tissue-originated spheroid (CTOS) lines derived from SCNEC of the uterine cervix using a method based upon cell-cell contact throughout the preparation and culturing processes. Using 11 CTOS lines, we assessed the sensitivity of various drugs used in clinical practice. Drug sensitivity assays revealed significant heterogeneous inter-CTOS chemosensitivity. Microarray analyses were then performed to identify sensitivity-related gene signatures. Specific gene sets were identified which likely contribute to the sensitivity to the tested drugs. We identified a line (Cerv54) that was exceptionally sensitive to irinotecan. Cerv54 had increased levels of CES1, which catalyzes the conversion of irinotecan to the active form, SN38, although in Cerv54 cells, SN38 was undetectable, CES1 expression and activity were markedly low compared to the liver, and a CES1 inhibitor had no effect on irinotecan sensitivity. These results suggested a novel irinotecan mode of action in Cerv54. Our CTOS lines may be useful for understanding the variation and mechanism of drug sensitivity, contributing to the understanding and development of chemotherapeutic drugs.

Small subset of Wnt-activated cells is an initiator of regrowth in colorectal cancer organoids after irradiation.

Most colorectal cancers (CRCs) are differentiated adenocarcinomas, which maintain expression of both stemness and differentiation markers. This observation suggests that CRC cells could retain a regeneration system of normal cells upon injury. However, the role of stemness in cancer cell regeneration after irradiation is poorly understood. Here, we examined the effect of radiation on growth, stemness, and differentiation in organoids derived from differentiated adenocarcinomas. Following a sublethal dose of irradiation, proliferation and stemness markers, including Wnt target genes, were drastically reduced, but differentiation markers remained. After a static growth phase after high dose of radiation, regrowth foci appeared; these consisted of highly proliferating cells that expressed stem cell markers. Radiosensitivity and the ability to form foci differed among the cancer tissue‐originated spheroid (CTOS) lines examined and showed good correlation with in vivo radiation sensitivity. Pre‐treating organoids with histone deacetylase inhibitors increased radiation sensitivity; this increase was accompanied by the suppression of Wnt signal‐related gene expression. Accordingly, Wnt inhibitors increased organoid radiosensitivity. These results suggested that only a small subset of, but not all, cancer cells with high Wnt activity at the time of irradiation could give rise to foci formation. In conclusion, we established a radiation sensitivity assay using CRC organoids that could provide a novel platform for evaluating the effects of radiosensitizers on differentiated adenocarcinomas in CRC.

Novel ex vivo disease model for extramammary Paget’s disease using the cancer tissue-originated spheroid method

BackgroundExtramammary Paget’s disease (EMPD) is a rare skin cancer that frequently occurs in the anogenital region in the elderly. Prognosis in patients with metastatic EMPD is poor as EMPD treatment has advanced little in recent years, primarily because no EMPD cell line has been established. ObjectiveWe aimed to establish an ex vivo EMPD disease model using the cancer tissue-originated spheroid (CTOS) method, which is used to prepare and culture primary cancer cells while maintaining cell–cell contact. MethodsThirteen samples from 12 EMPD patients were obtained. CTOSs were prepared and cultured using CTOS method. Histopathological examination of the CTOSs was performed. We investigated optimum medium conditions and effects of growth factors for CTOS growth. Chemo-sensitivity assays were conducted. ResultsCTOSs were successfully prepared from 3 primary lesions and 2 metastatic lymph nodes. Of these, 2 CTOSs (EMPD-3 and EMPD-4) could be maintained and passaged long term ex vivo. Following transplantation of CTOSs to NOD/Scid mice, CTOS-derived xenotumors exhibited ductal formation, indicating that CTOSs retained the original tumor characteristics. Chemo-sensitivity assays revealed that docetaxel significantly inhibited EMPD-3 growth in a dose-dependent manner, whereas EMPD-4 was not clearly inhibited. These findings indicate the heterogeneity of EMPD and potential use of chemosensitivity assays with patient-derived CTOS to select the most effective drugs for each patient. ConclusionTo our knowledge, this study represents the first establishment of an ex vivo-EMPD disease model involving conventional cell lines. EMPD CTOSs might be useful for developing new therapeutic strategies.

Dedifferentiation of neuroendocrine carcinoma of the uterine cervix in hypoxia

Neuroendocrine carcinoma of small cell type (SCNEC) is a rare pathological subtype in cervical cancer, which has a worse prognosis than other histological cell types. Due to its low incidence and the lack of experimental platforms, the molecular characteristics of SCNEC in the cervix remain largely unknown. Using the cancer tissue-originated spheroid (CTOS) method—an ex vivo 3D culture system that preserves the differentiation status of the original tumors—we established a panel of CTOS lines of SCNEC. We demonstrated that xenograft tumors and CTOSs, respectively, exhibited substantial intra-tumor and intra-CTOS variation in the expression levels of chromogranin A (CHGA), a neuroendocrine tumor marker. Since hypoxia affects differentiation in various tumors and in stem cells, we also investigated how hypoxia affected neuroendocrine differentiation of SCNEC of the uterine cervix. In the CTOS line cerv21, hypoxia suppressed expression of the neuroendocrine markers CHGA and synaptophysin (SYP). Flow cytometry analysis using CD99 (a membrane protein marker of SCNEC) revealed decreased CD99 expression in a subset of cells under hypoxic conditions. These expression changes were attenuated by HIF-1α knockdown, and by a Notch inhibitor, suggesting that these molecules played a role in the regulation of neuroendocrine differentiation. The examined SCNEC markers were suppressed under hypoxia in multiple CTOS lines. Overall, our present results indicated that neuroendocrine differentiation in SCNEC of the uterus is a variable phenotype, and that hypoxia may be one of the factors regulating the differentiation status.

Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers.

Resistance of progressive cancers against chemotherapy is a serious clinical problem. In this context, human epidermal growth factor receptor 3 (HER3) can play important roles in drug resistance to HER1- and HER2- targeted therapies. Since clinical testing of anti-HER3 monoclonal antibodies (mAbs) such as patritumab could not show remarkable effect compared with existing drugs, we generated novel mAbs against anti-HER3. Novel rat mAbs reacted with HEK293 cells expressing HER3, but not with cells expressing HER1, HER2 or HER4. Specificity of mAbs was substantiated by the loss of mAb binding with knockdown by siRNA and knockout of CRISPR/Cas9-based genome-editing. Analyses of CDR sequence and germline segment have revealed that seven mAbs are classified to four groups, and the binding of patritumab was inhibited by one of seven mAbs. Seven mAbs have shown reactivity with various human epithelial cancer cells, strong internalization activity of cell-surface HER3, and inhibition of NRG1 binding, NRG1-dependent HER3 phosphorylation and cell growth. Anti-HER3 mAbs were also reactive with in vivo tumor tissues and cancer tissue-originated spheroid. Ab4 inhibited in vivo tumor growth of human colon cancer cells in nude mice. Present mAbs may be superior to existing anti-HER3 mAbs and support existing anti-cancer therapeutic mAbs.

Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies

BackgroundSince the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC.MethodsWe performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened.ResultsDisulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA–platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin.ConclusionsThe present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.

Abstract 33: Ex vivo drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix

Abstract Small cell neuroendocrine carcinoma (SCNEC) of uterine cervix is a rare tumor accounting for approximately 1-2% of uterine cervical tumors. SCNEC of uterine cervix is highly invasive and has an extremely poorer prognosis than other histologic types for which no standard treatment is established. To establish a new management strategy against SCNEC of uterine cervix, we developed a drug sensitivity assay based on a 3D primary culture system. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method from patients’ tumors. To date, we established a panel of 12 SCNEC CTOS lines with the success rate of 100%. For each line, multiple CTOSs were placed in one well of a 96 well plate and dose response analysis was performed for seven anticancer drugs; paclitaxel, carboplatin, irinotecan, SN-38, cisplatin, etoposide and gemcitabine. After 7 days of culture, the CTOSs viability was evaluated by measuring intracellular ATP levels, corrected by the size of CTOS at day0. All drugs had substantial variations in sensitivity among the lines. Cerv54 was far more sensitive to irinotecan than other lines while was similarly sensitive to SN-38. We revealed that cancer cells of cerv54 had high levels of carboxylesterase(CES)1 expression, which converts irinotecan to SN38. Since cerv54 with CES1 knockdown by pGFP-C-shCES1 lentivirus had lower sensitivity than sh-scramble, CES1 expression is related to the irinotecan sensitivity in cerv54. CES1 expression in cancer cells may be useful for predicting the effect of irinotecan based chemotherapy. In addition, this study indicates that sensitivity test using CTOS might be applicable to precision medicine by selecting sensitive drugs for each patient. Citation Format: Mie Tanaka, Satoshi Kubota, Yu Ito, Yumiko Kiyohara, Hiroko Endo, Jumpei Kondo, Satoshi Nakagawa, Akiko Okazawa, Shinya Matsuzaki, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Shoji Kamiura, Tadashi Kimura, Masahiro Inoue. Ex vivo drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 33.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient‐derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high‐throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high‐throughput screening of 2427 drugs using the cancer tissue‐originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Spheroid Cultures of Primary Urothelial Cancer Cells: Cancer Tissue-Originated Spheroid (CTOS) Method.

Increasingly, it has been recognized that studying cancer samples from individual patients is important for the development of effective therapeutic strategies and in endeavors to overcome therapy resistance. Primary cultures of cancer cells acutely dissected from individual patients can provide a platform that enables the study and characterization of individual tumors. To that end, we have developed a method for preparing cancer cells in the form of multi-cellular spheroids. The cells can be derived from patient tumors (primary cells), from patient-derived xenografts, or from genetically- or chemically induced animal tumors. This method of culturing spheroids composed of cells derived from cancer tissues can be applied to various types of cancer, including urothelial cancer. The method is based on the principle of retaining cell-cell contact throughout cancer cell preparation and culturing. The first step is a partial digestion of the tumor specimen into small fragments; these fragments spontaneously form spheroidal shapes within several hours. The spheroid is referred to as a cancer tissue-originated spheroid (CTOS). The advantage of the CTOS method is that it allows one to prepare pure cancer cells at high yield. CTOSs can be stably cultured in serum-free conditions. The CTOS method can be applied to drug sensitivity assays, drug screening, and analyses of intracellular signaling. Moreover, the CTOS method provides a platform for studying the nature of cancer cell clusters.

In vivo and ex vivo cetuximab sensitivity assay using three-dimensional primary culture system to stratify KRAS mutant colorectal cancer.

In clinic, cetuximab, an anti-EGFR antibody, improves treatment outcomes in colorectal cancer (CRC). KRAS-mutant CRC is generally resistant to cetuximab, although difference of the sensitivity among KRAS-mutants has not been studied in detail. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method for cancer cells. We applied CTOS method to investigate whether ex vivo cetuximab sensitivity assays reflect the difference in sensitivity in the xenografts. Firstly, in vivo cetuximab treatment was performed with xenografts derived from 10 CTOS lines (3 KRAS-wildtype and 7 KRAS mutants). All two CTOS lines which exhibited tumor regression were KRAS-wildtype, meanwhile all KRAS-mutant CTOS lines grew more than the initial size: were resistant to cetuximab according to the clinical evaluation criteria, although the sensitivity was quite diverse. We divided KRAS-mutants into two groups; partially responsive group in which cetuximab had a substantial growth inhibitory effect, and resistant group which exhibited no effect. The ex vivo signaling assay with EGF stimulation revealed that the partially responsive group, but not the resistant group, exhibited suppressed ERK phosphorylation ex vivo. Furthermore, two lines from the partially responsive group, but none of the lines in the resistant group, exhibited a combinatory effect of cetuximab and trametinib, a MEK inhibitor, ex vivo and in vivo. Taken together, the results indicate that ex vivo signaling assay reflects the difference in sensitivity in vivo and stratifies KRAS mutant CTOS lines by sensitivity. Therefore, coupling the in vivo and ex vivo assays with CTOS can be a useful platform for understanding the mechanism of diversity in drug sensitivity.

Abstract 4254: Dynamic change of differentiation in spheroids derived from mixed small cell carcinoma/adenocarcinoma of the uterine cervix

Abstract Small cell carcinoma of the uterine cervix (SCCC) occurs mainly in young women with poor prognosis. Clear treatment algorithm remains difficult to develop for its rarity. Herein, we report a case of SCCC with adenocarcinoma component, and analyzed the mechanism of differentiation using a spheroid culture method. CTOS (cancer tissue-originated spheroid) is a primary culture system by which highly purified and viable cancer cells are cultivated. The principle of this method is maintaining cell-cell contact throughout the preparation and culture process. CTOS preserves characters of original tumors, especially their differentiation status. We generated a panel of CTOSs from 9 SCCCs. The success rate of CTOS preparation in SCCC was 100%, indicating SCCC is one of the most suitable cancers for CTOS method. We focused on SCCCs with adenocarcinoma component. These admixed tumors have been reported in other organs, such as lung, ovary, stomach, and so on. However, the mechanism underlying mixed tumor remains unclear. One of the CTOSs retained the characteristics of mixed tumor in original tumor. Immunostaining revealed that CD99 was positive in the region of small cell carcinoma region, and CK7 in that of adenocarcinoma. CTOSs were sorted by CD99 and EpCAM (a possible stem cell marker) expression levels, and transplanted into NOD-Scid mice. Xenotumors derived from both CD99lowEpCAMlow cells and CD99lowEpCAMhigh cells showed mixed cell type in H&E staining. Immunohistochemistry revealed that CD99 was positive in the region of small cell carcinoma, and CK7 in that of adenocarcinoma. On the other hand, CD99highEpCAMhigh cells-derived xenotumor consisted of only small cell carcinoma. The tumors were positive for CD99, but negative for CK7. Next, we searched for the key factor of differentiation. It has been reported that hypoxia induced adenocarcinoma in lung adenosquamous cell carcinoma. When CTOSs were incubated in hypoxia (3%O2), FACS analysis revealed that proportion of CD99low cells increased (20.6% in normoxia, 40.2% in hypoxia). Immunostaining of CTOSs showed similar results. Thus differentiation status of a mixed type SCCC showed dynamic change of differentiation in spheroids depending on culture conditions. The research can provide insight into the origin of mixed tumor of SCCC, and may be useful for developing treatment strategy. Citation Format: Satoshi Kubota. Dynamic change of differentiation in spheroids derived from mixed small cell carcinoma/adenocarcinoma of the uterine cervix. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4254.

Abstract 3341: Microarray analysis of gastrointestinal stromal tumor-originated spheroids

Abstract Background: Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit are thought to occur during tumor progression. Although sequential therapy with tyrosine kinase inhibitors (TKIs), imatinib mesylate, sunitinib malate and regorafenib, is an effective and standard treatment strategy for recurrent or metastatic GISTs, it is difficult to cure recurrent GISTs with TKIs alone. It has been reported that viable cells are often seen in tumors resected in response to imatinib therapy and they may cause refractory tumors after interruption of imatinib in responding patients with advanced GISTs. Therefore targeting these persistent clones, which may have stem cell characteristics, can be a novel treatment strategy to cure recurrent or metastatic GISTs. In this study, we generated spheroids from resected GIST and performed microarray analysis to identify gene sets typically expressed in GIST-originated spheroid (GISTOS). Methods: According to the previous report of cancer tissue originated spheroid (CTOS) (Kondo J et al. PNAS 108:6235-40, 2011), GISTOS was prepared from fresh samples of 6 primary gastric GISTs. Total RNAs were extracted from 6 GISTOS and their original tumors and utilized for cDNA microarray analysis. None of the patients had received imatinib therapy before surgery. Gene expression levels were compared between GISTOSs and their original tumors. Results: Hierarchical clustering revealed two distinct clusters, and distinguished GISTOS from original tumors. Gene set enrichment analysis (GSEA) revealed that the gene sets CISPLATIN_RESISTSNCE, METASTASIS_STROMAs and TUMOR_INVASIVENESS_2D were upregulated and gene sets CELL_CYCLE_G1_S and MIR21_TARGETS were downregulated in GISTOSs compared with original tumors. These gene sets appear to reflect distinctive features of GISTOS that consists of enriched stem cell populations. Conclusions: We generated GIST-originated spheroids. Microarray analysis revealed gene sets up-regulated and down-regulated in GISTOS. These gene sets appear to reflect distinctive features of GISTOS and may serve as novel molecular markers of stem cells in GIST. Citation Format: Hirotoshi Kikuchi, Shinichiro Miyazaki, Tomohiro Murakami, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Manabu Ohta, Takanori Sakaguchi, Hiroyuki Konno. Microarray analysis of gastrointestinal stromal tumor-originated spheroids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3341.

Corrigendum: Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen.

Scientific Reports 6: Article number: 24823; published online: 21 April 2016; updated: 31 May 2016 This Article contains a typographical error in the Results section under subheading ‘5G2 mAb recognized sugar chain epitopes’, “The 5G2 mAb recognized the Lea (Galβ1-3(Fucα1-2)GlcNAc), Lec (Galβ1-3GlcNAc), core1 (Galβ1-3GalNAc), core2 (Galβ1-3(GlcNAcβ1-6)GalNAc), and Siaα2-6core1 (Siaα2-6(Galβ1-3)GalNAc) structures (Fig.

Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen.

Spheroids cultured directly from tumours can better reflect in vivo tumour characteristics than two-dimensional monolayer culture or three-dimensional culture of established cell lines. In this study, we generated antibodies by directly immunizing mice with primary-cultured living spheroids from human colorectal cancer. We performed phenotypic screening via recognition of the surface of the spheroids and inhibition of their adhesion to extracellular matrices to identify a monoclonal antibody, clone 5G2. The antibody inhibited cell migration in two-dimensional culture and promoted cell detachment. Western blotting and immunohistochemistry detected the 5G2 signal in many colorectal cancer spheroids, as well as patient tumours, but failed to detect in various cell lines examined. We found that 5G2 recognized the Lea and Lec on N-glycan, and their major carrier proteins were CEACAM5 and CEACAM6. Pre-incubation of the spheroids with 5G2 impaired translocation of integrin β4 from the lateral membrane to the contact interface between the extracellular matrix when embedded in it. As we successfully obtained a functional antibody, which antigen was glycan structures and lost in cell lines, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies.