Growth Of Epithelial Tumor Cells Research Articles

An Effective Primary Head and Neck Squamous Cell Carcinoma In Vitro Model.

Head and neck squamous cell carcinoma is a highly malignant disease and research is needed to find new therapeutic approaches. Faithful experimental models are required for this purpose. Here, we describe the specific cell culture conditions enabling the efficient establishment of primary cell culture models. Whereas a classical 10% serum-containing medium resulted in the growth of fibroblast-like cells that outcompeted epithelial cells, we found that the use of specific culture conditions enabled the growth of epithelial tumor cells from HPV+ and HPV− head and neck cancer tissue applicable for research. EpCAM and high Thy-1 positivity on the cell surface were mutually exclusive and distinguished epithelial and fibroblast-like subpopulations in all primary cultures examined and thus can be used to monitor stromal contamination and epithelial cell content. Interestingly, cells of an individual patient developed tumor spheroids in suspension without the use of ultra-low attachment plates, whereas all other samples exclusively formed adherent cell layers. Spheroid cells were highly positive for ALDH1A1 and hence displayed a phenotype reminiscent of tumor stem cells. Altogether, we present a system to establish valuable primary cell culture models from head and neck cancer tissue at high efficiency that might be applicable in other tumor entities as well.

P3.02-073 Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma

Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+; Trp53fl/fl(KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization and qPCR that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. The Hh signaling pathway acts upon lung stromal cells in a paracrine fashion and induces distinct transcriptional programs in murine embryonic and lung fibroblasts. Inhibition of paracrine Hh pathway activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung adenocarcinomas express high levels of IHH that dominates the tumor suppressive effects in our mouse models.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90−, CD44−/CD90−, CD44+/CD90+, CD44−/CD90−). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90−) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44− subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44− subpopulations. Furthermore, co-cultivation experiments revealed that CD44−/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90−). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44−/CD90+, but this subpopulation had a low migratory capacity.

Abstract 2964: Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung tumor epithelia. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss,, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+;Trp53fl/fl (KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. In conclusion, the Hh signaling pathway acts upon lung stromal cells in a paracrine fashion. Inhibition of Hh activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung cancers express high levels of IHH that dominates the tumor suppressive effect of our mouse models. Citation Format: Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Umaru Barrie, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2017-2964

Abstract 3303: Identifying a functional correlation between cancer stem cells and 3D clonogenic growth: testing rational combinations of PI3K pathway inhibitors in subtype-specific BC

Abstract Introduction: Studying clonogenic behavior of tumor cells in the context of physiologically relevant ECM offers the potential to explore signaling processes which more closely approximate the in vivo microenvironment. This approach is valuable for identification and dissection of the key molecular signaling of cancer cells to test targeted drugs. 3D cell cultures have been recognized as the method of choice for the physiologically relevant modeling of many aspects of malignant cell behavior ex vivo. Kenny et al., 2007 identified that morphologies of breast cancer cell lines in 3D assays correlate with their gene expression profiles and protein expression patterns reflecting the underlying distinct morphologies which were associated with tumor cell invasiveness and metastases-associated phenotypes. Study of the molecular characteristics and clinical significance of CSC in the context of prognostic relevance as well as the understanding the scope of therapeutically targeting CSC within distinct tumor cell populations have revealed that CD44+ cell-specific genes represents SC markers and correlated with decreased patient survival (Fillmore and Kuperwasser, 2008). Hypothesis: Since 3D clonogenic growth of epithelial tumor cells represents the most appropriate model in vitro, we hypothesized that there is a functional association between phenotypic 3D clonogenic growth and cancer stem cells (CSC) proportion. Methods: To study the role of CSCs in the 3D clonogenic growth of tumor cells we have used BC and ovarian cell lines.The cells were challenged to form 3D colonies in matrigel and cells were recovered from the 3D colonies after de-gelling using PBS-EDTA buffer. Relative distribution of CD24+, CD44+, CD24- and CD44- expression in cells were determined by flow cytometry (Accuri C6) following recovery of the cells from the colony and compared to cells grown in 2D. We have tested the effect of a rational combination of pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells. Results: CD44/CD24 expression level changes between 2D and 3D growth was characterized in individual cell lines. Overall 3D growth led to a reduction in CD44 levels. Rational drug combinations blocked proliferative signals and enhanced apoptosis (cleaved caspase3) as demonstrated by WB, AnnexinV staining, proliferation (Incucyte). The mechanistic details of the effect of this combination on the CD44H/CD24L stem cell populations are being worked out, the results of which will be presented in the meeting. Significance: Our study reports a methodological breakthrough to determine CD24L/CD44H CSC fraction from live cells following 3D clonogenic transformation. Citation Format: Jennifer H. Carlson, Nandini Dey, Pradip De, Lori Friedman, Casey Williams, Brian Leyland-Jones. Identifying a functional correlation between cancer stem cells and 3D clonogenic growth: testing rational combinations of PI3K pathway inhibitors in subtype-specific BC. [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 3303.

Stress-induced Premature Senescence (SIPS)

Replicative senescence is a fundamental feature in normal human diploid cells and results from dysfunctional telomeres at the Hayflick cell division limit. Ionizing radiation (IR) prematurely induces the same phenotypes as replicative senescence prior to the Hayflick limit. This process is known as stress-induced premature senescence (SIPS). Since the cell cycle is irreversibly arrested in SIPS-induced cells, even if they are stimulated by various growth factors, it is thought that SIPS is a form of cell death, irreversibly eliminating replicating cells. IR-induced-focus formation of DNA repair proteins, a marker of DNA damage, is detected in SIPS as well as replicative senescent cells. Furthermore, both processes persistently induce cell cycle checkpoint mechanisms, indicating DNA damage created by ionizing radiation induces SIPS in normal cells, possibly by the same mechanisms as those occurring in replicative senescence. Interestingly, IR induces SIPS not only in normal cells, but also in tumor cells. Due to the expression of telomerase in tumor cells, telomere-dependent replicative senescence does not occur. However, SIPS is induced under certain conditions after IR exposure. Thus, cell death triggered by IR can be attributed to apoptosis or SIPS in tumor cells. However, metabolic function remains intact in SIPS-induced cancer cells, and recent studies show that senescence eliminate cells undergoing SIPS secrete various kinds of factors outside the cell, changing the microenvironment. Evidence using co-culture systems containing normal senescent stromal cells and epithelial tumor cells show that factors secreted from senescent stroma cells promote the growth of tumor epithelial cells both in vitro and in vivo. Thus, regulation of factors secreted from SIPS-induced stromal cells, as well as tumor cells, may affect radiotherapy.

TGFβ signaling is necessary for carcinoma cell invasiveness and metastasis

Background: Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor β (TGFβ) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. Many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFβ, however, and even secrete TGFβ. In line with this, TGFβ is involved in angiogenesis, wound healing and epithelial–mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFβ-induced EMT maintained via a TGFβ autocrine loop. Thus, we have analyzed whether signal transduction by the TGFβ receptor (TGFβR) is required for local tumor cell invasion and metastasis.Results: A dominant-negative type II TGFβR (TGFβRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFβRII-dn blocked TGFβR signaling and heavily delayed tumor formation. In EpRas cells, TGFβRII-dn prevented EMT. In the dedifferentiated mesenchymal CT26 cells, TGFβRII-dn caused mesenchymal-to-epithelial transition and inhibited their in vitro invasiveness in several assays. In addition, TGFβRII-dn completely abolished metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lost in vitro invasiveness when treated with neutralizing TGFβ antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFβRII were non-invasive in vitro, a defect restored by re-expressing wild-type TGFβRII.Conclusions: Cell-autonomous TGFβ signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFβRII therefore represents a potential target for therapeutical intervention in human tumorigenesis.

Heparin-binding growth factor/prostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta.

Normal rat prostate epithelial cell growth requires both epidermal growth factor and heparin-binding growth factor/prostatropin. In contrast, epithelial cells derived from the transplantable Dunning R3327H rat tumor require either epidermal growth factor or heparin-binding growth factor/prostatropin. Transforming growth factor type beta inhibited normal epithelial cell growth. Transforming growth factor beta inhibited epidermal growth factor-dependent growth of tumor epithelial cells, independent of epidermal growth factor concentrations. Transforming growth factor beta increased the effective dose of heparin-binding growth factor type 1 required to support tumor epithelial cell growth by 10-fold but saturating levels of heparin-binding growth factor type 1 (290 pM) completely attenuated the inhibitory effect of transforming growth factor beta. These results suggest that prostate tumor epithelial cells may escape the inhibitory effect of transforming growth factor beta as a consequence of alteration of the concurrent requirement for both epidermal growth factor (or homologues) and heparin-binding growth factors.

Autocrine growth factors for human tumor clonogenic cells.

A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.