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Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment.

ABSTRACT: Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.

SUBMITTER: Daster S

PROVIDER: S-EPMC5352092 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment.

Däster Silvio S Amatruda Nunzia N Calabrese Diego D Ivanek Robert R Turrini Eleonora E Droeser Raoul A RA Zajac Paul P Fimognari Carmela C Spagnoli Giulio C GC Iezzi Giandomenica G Mele Valentina V Muraro Manuele G MG

Oncotarget 20170101 1

Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles i ...[more]

PMID: 27965457

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Project description:PurposeUveal melanoma (UM) has a high propensity for metastatic spread, and approximately 40-50% of patients die of metastatic disease. Metastases can be found at the time of diagnosis but also several years after the tumor has been removed. The survival of disseminated cancer cells is known to be linked to anchorage independence, anoikis resistance, and an adaptive cellular metabolism. The cultivation of cancer cells as multicellular tumor spheroids (MCTS) by anchorage-independent growth enriches for a more aggressive phenotype. The present study examines the differential gene expression of adherent cell cultures, non-adherent MCTS cultures, and uncultured tumor biopsies from three patients with UM. We elucidate the biochemical differences between the culture conditions to find whether the culture of UM as non-adherent MCTS could be linked to an anchorage-independent and more aggressive phenotype, thus unravelling potential targets for treatment of UM dissemination.MethodsThe various culture conditions were evaluated with microarray analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNAscope, immunohistochemistry (IHC), and transmission electron microscopy (TEM) followed by gene expression bioinformatics.ResultsThe MCTS cultures displayed traits associated with anoikis resistance demonstrated by ANGPTL4 upregulation, and a shift toward a lipogenic profile by upregulation of ACOT1 (lipid metabolism), FADS1 (biosynthesis of unsaturated fatty acids), SC4MOL, DHCR7, LSS (cholesterol biosynthesis), OSBPL9 (intracellular lipid receptor), and PLIN2 (lipid storage). Additionally, the present study shows marked upregulation of synovial sarcoma X breakpoint proteins (SSXs), transcriptional repressors related to the Polycomb group (PcG) proteins that modulate epigenetic silencing of genes.ConclusionsThe MCTS cultures displayed traits associated with anoikis resistance, a metabolic shift toward a lipogenic profile, and upregulation of SSXs, related to the PcG proteins.

Project description:BackgroundLung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies.MethodsTo overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy.ResultsWe showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level.ConclusionsThis study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

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Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment.

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Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment.

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