Tumor Fragment Spheroids Research Articles

Autophagy in 3D In Vitro and Ex Vivo Cancer Models.

Three-dimensional (3D) models are acquiring importance in cancer research due to their ability to mimic multiple features of the tumor microenvironment more accurately than standard monolayer two-dimensional (2D) cultures. Several groups, including our laboratory, are now accumulating evidence that autophagy in solid tumors is also better represented in 3D than in 2D. Here we detail how we generate 3D models, both in vitro multicellular spheroids generated from cell lines and ex vivo tumor fragment spheroids generated from tumor samples, and how autophagy can be measured in 3D cultures.

Inhibition of autophagy initiation potentiates chemosensitivity in mesothelioma.

The benefits of inhibiting autophagy in cancer are still controversial, with differences in outcome based on the type of tumor, the context and the particular stage of inhibition. Here, we investigated the impact of inhibiting autophagy at different stages on chemosensitivity using 3-dimensional (3D) models of mesothelioma, including ex vivo human tumor fragment spheroids. As shown by LC3B accumulation, we successfully inhibited autophagy using either an early stage ULK1/2 inhibitor (MRT 68921) or a late stage inhibitor (hydroxychloroquine). We found that inhibition of autophagy at the early stage, but not at late stage, potentiated chemosensitivity. This effect was seen only in those spheroids with high autophagy and active initiation at steady state. Inhibition of autophagy alone, at either early or late stage, did not cause cell death, showing that the inhibitors were non-toxic and that mesothelioma did not depend on autophagy at baseline, at least over 24 h. Using ATG13 puncta analysis, we found that autophagy initiation identified tumors that are more chemosensitive at baseline and after autophagy inhibition. Our results highlight a potential role of autophagy initiation in supporting mesothelioma cells during chemotherapy. Our work also highlights the importance of testing the inhibition of different stages in order to uncover the role of autophagy and the potential of its modulation in the treatment of cancer.

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

ABSTRACTUnderstanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.

Autophagy Correlates with the Therapeutic Responsiveness of Malignant Pleural Mesothelioma in 3D Models.

Malignant pleural mesothelioma is a highly chemoresistant solid tumor. We have studied this apoptotic resistance using in vitro and ex vivo three-dimensional models, which acquire a high level of chemoresistance that can be reduced by PI3K/mTOR inhibitors. Here, we investigate the activity of GDC-0980, a novel dual PI3K/mTOR inhibitor, which has been proposed to be effective in mesothelioma. In this work, we aimed to identify mechanisms and markers of efficacy for GDC-0980 by utilizing 3D models of mesothelioma, both in vitro multicellular spheroids and ex vivo tumor fragment spheroids grown from patient tumor samples. We found that a subset of mesothelioma spheroids is sensitive to GDC-0980 alone and to its combination with chemotherapy. Unexpectedly, this sensitivity did not correlate with the activation of the Akt/mTOR pathway. Instead, sensitivity to GDC-0980 correlated with the presence of constitutive ATG13 puncta, a feature of autophagy, a cellular program that supports cells under stress. In tumor fragment spheroids grown from 21 tumors, we also found a subset (n = 11) that was sensitive to GDC-0980, a sensitivity that also correlated with the presence of ATG13 puncta. Interference with autophagy by siRNA of ATG7, an essential autophagic protein, increased the response to chemotherapy, but only in the sensitive multicellular spheroids. In the spheroids resistant to GDC-0980, autophagy appeared to play no role. In summary, we show that GDC-0980 is effective in mesothelioma 3D models that display ATG13 puncta, and that blockade of autophagy increases their response to chemotherapy. For the first time, we show a role for autophagy in the response to chemotherapy of 3D models of mesothelioma and propose ATG13 as a potential biomarker of the therapeutic responsiveness of mesothelioma.

Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.

Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2–3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

Abstract LB-251: Manipulation of macrophage phenotype enhances the apoptotic response to chemotherapy in mesothelioma

Abstract Macrophages within the tumor microenvironment (TME) may contribute to tumor chemoresistance. We have found that macrophages constitute a significant percentage of the infiltrating leukocytes in mesothelioma, a highly chemoresistant tumor. We asked whether reprogramming of macrophage phenotype from a Th2 (pro-tumor) to a Th1 (anti-tumor) phenotype would alter the mesothelioma chemoresponsiveness. To address this, we used 3D organotypic models of mesothelioma growth: 1) multicellular spheroids, with mesothelioma cells grown alone or co-cultured with macrophages derived from peripheral blood monocytes, and 2) primary tumor fragment spheroids derived from small fragments of freshly isolated human mesothelioma. Macrophages were incubated with Th1 (LPS & IFNγ) or Th2 (IL-4 &IL-13) cytokines prior to co-culture with mesothelioma spheroids, followed by exposure to standard-of-care chemotherapy, cisplatin plus pemetrexed. Gene expression was evaluated in macrophages to affirm Th1-type (TNF, IL-12, IFNα) versus Th2-type (CD206, IL10) programming. In both spheroid approaches, Th1-type macrophage programming significantly increased the apoptotic response of mesothelioma cells to chemotherapy. Moreover, when CSF1R signaling in macrophages was inhibited by incubation with a small molecular weight kinase antagonist, GW2850, chemoresponsiveness was significantly increased as evidenced by increased presence of apoptotic tumor cells in spheroids. Enhanced chemoresponsiveness of mesothelioma tumor cells was dependent on presence of macrophages because incubation of tumor cells with Th1-type cytokines or GW2850 in the absence of macrophages was without effect. We conclude that manipulating the TME may be a promising therapeutic approach in mesothelioma. This work was supported by a DOD Mesothelioma Program grant PR80717 to Broaddus and Coussens and a T32 Training Grant to Battula and Blakely. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-251. doi:1538-7445.AM2012-LB-251

Identification of MCAM/CD146 as the Target Antigen of a Human Monoclonal Antibody that Recognizes Both Epithelioid and Sarcomatoid Types of Mesothelioma

The prognosis for patients diagnosed with mesothelioma is generally poor, and currently available treatments are usually ineffective. Therapies that specifically target tumor cells hold much promise for the treatment of cancers that are resistant to current approaches. We have previously selected phage antibody display libraries on mesothelioma cell lines to identify a panel of internalizing human single chain (scFv) antibodies that target mesothelioma-associated, clinically represented cell surface antigens and further exploited the internalizing function of these scFvs to specifically deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells. Here, we report the identification of MCAM/MUC18/CD146 as the surface antigen bound by one of the mesothelioma-targeting scFvs using a novel cloning strategy based on yeast surface human proteome display. Immunohistochemical analysis of mesothelioma tissue microarrays confirmed that MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma tumor cells in situ but not by normal mesothelial cells. In addition, quantum dot-labeled anti-MCAM scFv targets primary meosthelioma cells in tumor fragment spheroids cultured ex vivo. As the first step in evaluating the therapeutic potential of MCAM-targeting antibodies, we performed single-photon emission computed tomography studies using the anti-MCAM scFv and found that it recognizes mesothelioma organotypic xenografts in vivo. The combination of phage antibody library selection on tumor cells and rapid target antigen identification by screening the yeast surface-displayed human proteome could be a powerful method for mapping the targetable tumor cell surface epitope space.

MTOR Mediates Survival Signals in Malignant Mesothelioma Grown as Tumor Fragment Spheroids

Solid tumors such as mesothelioma exhibit a stubborn resistance to apoptosis that may derive from survival pathways, such as PI3K/Akt/mTOR, that are activated in many tumors, including mesothelioma. To address the role of PI3K/Akt/mTOR, we used a novel approach to study mesothelioma ex vivo as tumor fragment spheroids. Freshly resected mesothelioma tissue from 15 different patients was grown in vitro as 1- to 2-mm-diameter fragments, exposed to apoptotic agents for 48 hours with or without PI3K/Akt/mTOR inhibitors, and doubly stained for cytokeratin and cleaved caspase 3 to identify apoptotic mesothelioma cells. Mesothelioma cells within the tumor spheroids exhibited striking resistance to apoptotic agents such as TRAIL plus gemcitabine that were highly effective against monolayers. In a majority of tumors (67%; 10 of 15), apoptotic resistance could be reduced by more than 50% by rapamycin, an mTOR inhibitor, but not by LY294002, a PI3K inhibitor. Responsiveness to rapamycin correlated with staining for the mTOR target, p-S6K, in the original tumor, but not for p-Akt. As confirmation of the role of mTOR, siRNA knockdown of S6K reproduced the effect of rapamycin in three rapamycin-responsive tumors. Finally, in 37 mesotheliomas on tissue microarray, p-S6K correlated only weakly with p-Akt, suggesting the existence of Akt-independent regulation of mTOR. We propose that mTOR mediates survival signals in many mesothelioma tumors. Inhibition of mTOR may provide a nontoxic adjunct to therapy directed against malignant mesothelioma, especially in those with high baseline expression of p-S6K.

Inhibition of the PI3K and mTOR survival pathways enhances apoptosis in human mesothelioma tumor fragment spheroids

Malignant mesothelioma exhibits significant resistance to apoptosis. We have characterized a novel in vitro model of human mesothelioma tissue grown in culture as tumor fragment spheroids to identify the mechanisms of resistance. Spheroids maintained many features of the original tumors, including the presence of viable proliferating mesothelioma cells, for up to 3 months. Mesothelioma cell monolayers or 2-week old human mesothelioma spheroids were exposed to the death receptor ligand TRAIL and cycloheximide (CHX) for 48 h and studied for apoptosis by expression of cleaved caspase 3 in mesothelioma (cytokeratin-positive) cells. Unlike the nearly 100% apoptosis in the monolayers, TRAIL plus CHX induced apoptosis in only 32 ± 4% (mean ± SEM) of mesothelioma cells within spheroids (versus 7 ± 1% in untreated spheroids). Apoptosis of mesothelioma cells in spheroids increased to 47 ± 6% by inhibition of the PI3K pathway with LY294002 and to 50 ± 17% by inhibition of the mTOR pathway with rapamycin. Inhibition of the pathways was confirmed with staining for p-Akt or p70-S6K, targets of the PI3K and mTOR pathways, respectively. Targeting these survival pathways therefore presents a promising approach for mesothelioma and perhaps other tumors resistant to apoptosis. This research was supported by an NIH R01 NCI-095671 to VCB.

A Novel In Vitro Model of Human Mesothelioma for Studying Tumor Biology and Apoptotic Resistance

Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagen-rich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 +/- 6% apoptosis; mean +/- SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 +/- 4% apoptosis; mean +/- SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 +/- 6% apoptosis) or the mTOR pathway with rapamycin (50 +/- 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy.

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

Progesterone receptors in tumor fragment spheroids of human meningiomas.

Progesterone receptors (PgR) are detectable in about 60-70% of tissue specimens of human meningiomas. Despite these data, PgR are hardly to be found in monolayer tissue culture of meningiomas. Aim of this study was to elucidate whether PgR might be preserved in tumor fragment spheroids of meningiomas maintained in organ culture since the morphological appearance of the original tumor is preserved by this culture technique. Aliquots of meningioma specimens of 25 patients (17 females) were snap frozen in liquid nitrogen immediately after removal. Additionally, monolayer tissue cultures of the same specimen were obtained as primary culture and passage #3. Tumor fragment spheroids were kept on medium-agar with liquid medium overlay and harvested after 1 and 3 weeks in culture. PgR were detected by immunohistochemistry using a rat monoclonal antibody. 18/25 meningioma tissue specimens were positive for PgR. In 8 out of 15 PgR-positive tumors which formed spheroids we could detect PgR in fragment spheroids after 1 and 3 weeks in culture. In contrast, none of the monolayers depicted PgR. PgR is preserved in a considerable amount of tumor fragment spheroids of PgR-positive meningiomas. They remain detectable after 3 weeks of culture whereas monolayer tissue cultures are PgR-negative. Thus, tumor fragment spheroids seem to be a suitable tool to investigate progesterone/antiprogesterone effects in vitro.