3D Clonogenic Assay Research Articles

Abstract P082: Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays

Abstract Plinabulin (Plin) is a new chemical entity and a selective immunomodulating microtubule-targeting agent (SIMBA), which exerts immune-enhancing effects through increasing dendritic cell (DC) maturation and DC-dependent antigen presentation to CD4+ and CD8+ T-Cells. Here we evaluated whether Plin also exerts direct anti-cancer effects against approximately 80 patient derived (PDX) models. Plin was characterized for its ability to inhibit anchorage independent growth and ex vivo colony formation of PDX tumor cells in semi-solid medium. The compound was investigated in PDX models of various cancer types, using a 3D clonogenic assay in a 96-well format. Briefly, tumor cell suspensions were prepared from subcutaneous xenografts in NMRI nu/nu mice and seeded into 96 well ultra-low attachment plates within a layer of semi-solid medium. Plin was added to the assay plate at either day 2 or day 7 and tested at 9 concentrations in half-log increments up to a concentration of 3 µM. Supernatant was exchanged 24 h after dosing and cells were further incubated at 37°C and 7.5 % CO2 up to a total assay time of 8 or 13 days. Colony counts based on image analysis were utilized to evaluate efficacy. Plin inhibited tumor colony formation in a concentration-dependent manner. With Plin treatment beginning 2 days after PDX cell seeding, the most sensitive cancer types were small cell lung cancer (mean absolute IC70 = 0.035 μM; n = 7), bladder cancer (mean absolute IC70 = 0.038 μM; n = 9), and soft tissue sarcoma (mean absolute IC70 = 0.057 μM; n = 10). Melanoma models were observed to be the least sensitive to Plin monotherapy, with a mean absolute IC70 of 1.105 μM (n = 19). With Plin treatment beginning 7 days after PDX cell seeding, based on absolute IC70 values, 9 out of 68 tumor models were sensitive towards plinabulin. The most sensitive histotypes were gastric cancer (Asian, mean abs. IC70 = 0.319 µM, n = 3), small cell lung cancer (mean abs. IC70 = 0.385 µM, n = 7; 3 below 50 nM), osteosarcoma (mean abs. IC70 = 0.624 µM, n = 3), and central nervous system cancer (mean abs. IC70 = 1.521 µM, n = 6). Overall, the most responsive models based on IC70 were the small cell lung cancer models 2156 (abs. IC70 = 0.015 µM), 1129 (abs. IC70 = 0.032 µM) and 650 (abs. IC70 = 0.032 µM), and the osteosarcoma model 1186 (abs. IC70 = 0.027 µM). Soft tissue sarcoma models (n = 8), Her2-enriched breast cancer models (n = 6), melanoma models (n = 9), bladder cancer models (n = 6), and gastric cancer models (Caucasian, n = 3) were observed to be the least sensitive cancer types with a mean IC70 value > 3 µM. In conclusion, using patient-derived tumor models in a 3D clonogenic assay, SCLC tumor types were the most sensitive to Plin. These results provide mechanistic support for the preliminary positive results recently reported with Plin in combination with Nivolumab and Ipilimumab in SCLC patients (Malhotra ASCO 2021). Citation Format: George K. Lloyd, James Tonra, Claudia Goettlich, Tobias Deigner, Ramon Mohanlal, Lan Huang. Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P082.

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma.

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.

Abstract 487: Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy

Abstract Background: Mesenchymal stem cells (MSCs) have the potential as a drug delivery tool in cancer immunotherapy. We previously demonstrated that MSCs can be used to deliver BCG immunotherapy to the hypoxic core of tumors (#AACR-Abstract-3903). However, lack of clinical grade MSCs hamper the use of MSCs in clinical practice. The production of clinical grade human derived MSCs in accordance with current Good Manufacture Practice (cGMP) is an unmet need in the cancer immunotherapy. However, the selection criterion for characterizing the safety of MSC-based products is not well cited yet. In an effort to generate a potent therapeutic cell based product, we design a series of in-vitro safety assays for the production of high quality tissue specific human MSCs (hMSCs) as a final products before releasing from cGMP. Method: Here, we have isolated MSCs from Bone marrow (BM), Adipose tissue (AD) and Wharton Jelly (WJ) and expanded in serum free media. The in-vitro proliferation and phenotypic profiling of stem cell markers performed to check purity of the ex-vivo cultured hMSCs. To determine the clinical safety, we have done stemness marker analysis and cellular senescence capacity. To determine the functional aspects, tri-lineage differentiation and immunopotency of hMSCs were performed. Additionally, to assure the in vitro anti-tumorigenic properties of characterized hMSCs subjected to 2D colony forming unit (CFU) assay, 3D clonogenic assay as 3D organoid culture system and karyotyping. Results: Here, we proposed bioprocess design that allowed successful isolation of MSCs from BM, AD and WJ separately. Growth profiles of all isolated MSCs (n = 3) under serum free condition media with respect to serum containing media shows an exponential growth phase. The decline phase started after Day 7. Results also shows significant variation in population doubling time (PDT) of all isolated hMSCs at Passage 3 (n = 3) and Passage 5((n = 3) in comparison between serum free condition media & serum containing media. We found all hMSCs cultured in serum free media shows positive for CD105-APC (>80%), CD73-PE (>80%), CD90- PerCP (>80%), CD29- FITC (>80%) and HLAI (>80%), and negative HLAII- FITC(<2.20 %) and CD34/45- FITC (<2.20 %) expressions at Passage 3 & passage 5, indicating a stem cell phenotype (n = 3). Isolated hMSCs showed osteocyte, adipocytes and chondrocytes differentiation and high capacity to inhibit the proliferation of activated lymphocytes. Additionally, hMSCs positively expressed Nanog and Sox-2 stemness gene and cellular senescence from passage 7. In 2D CFU assay these cells showed colony forming capacity and in 3D organoid culture system in soft agar based matrix, we found that these cells are non-tumorigenic and didn't showed any karyotypic alteration. Conclusion: The collective result provides compelling evidence of the clinical safety of cGMP-compliant BM-MSCs, AD-MSCs and WJ-MSCs potentially suitable for future therapeutic application. Citation Format: Sorra Sandhya, Sonali Rawat, Suchi Gupta, Harshita Sharma, Bikul Das, Sujata Mohanty. Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 487.

In vitro therapy against glioblastoma cells by 3-Dezaneplanocin-A, panobinostat, and temozolomide

Background: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current treatment against this tumor consists of maximal surgical resection without threatening the patient's life, followed by a treatment with temozolomide, with or without combined radiotherapy. GBM is resistant to the conventional antitumor therapies, so in this research, we tried to inhibit tumor growth with the combination of three drugs: (1) panobinostat, an inhibitor of histone deacetylases, (2) 3-Dezaneplanocin-A (DZNep), an inhibitor of EZH2, a protein which belongs to the polycomb repressor complex 2, acting as a histone methylase, and (3) temozolomide, an alkylating agent. Methods: The T98G GBM commercial cell line was used. Cells were exposed to single treatments of the drugs and to the three possible combinations among them. Soon after, two-dimensional (2D) and 3D clonogenic assays were assessed for in vitro tumorigenicity testing. Real-time quantitative polymerase chain reaction of 2 proapoptotic genes (BAX and NOXA) and 2 antiapoptotic genes (BCL2 and BCL-XL) was also assessed. Results: The panobinostat and temozolomide combination produced a positive effect against T98G glioblastoma cells by reducing soft agar colony formation, by inducing high expression levels of NOXA, and by reducing BCL-XL expression. Equally, the panobinostat and DZNep combination produced a positive effect against T98G glioblastoma cells by reducing colony formation in adherent conditions and by inducing high expression levels of BAX. Finally, temozolomide alone was the most efficient drug for decreasing BCL2 expression. Conclusion: Panobinostat and temozolomide combination or panobinostat and DZNep combination might be more efficient against glioblastoma cells than just temozolomide.

Discovery of potent and selective cytotoxic activity of new quinazoline-ureas against TMZ-resistant glioblastoma multiforme (GBM)

Herein, we report new quinazoline-urea based compounds with potent cytotoxic activities against TMZ-resistant glioblastoma multiforme (GBM) cells. Low micromolar IC50 values were exhibited over a panel of three primary GBM patient-derived cell cultures belonging to proneural (GBM-1), mesenchymal (GBM-2), and classical (GBM-3) subtypes. Eight compounds showed excellent selectivity indices for GBM cells comparing to a normal astrocyte cell line. In JC-1 assay, analogues 11, 12, 20, 22, and 24 exerted promising rates of mPTP opening induction towards proneural GBM subtype. Compounds 11, 20, and 24 bound to the translocator protein 18 kDa (TSPO) in submicromolar range using [3H] PK-11195 binding affinity assay. A homology model was built and docked models of 11, 12, 20, 22 and 24 were generated for describing their plausible binding modes in TSPO. In 3D clonogenic assay, compound 20 manifested potent tumoricidal effects on TMZ-resistant GBM cells even at submicromolar concentrations. In addition, CYP450 and hERG assays presented a safe toxicity profile of 20. Taken as a whole, this report presents compound 20 as a potent, selective and safe GBM cytotoxic agent which constitutes a promising direction against TMZ-resistant GBM.

Abstract 2561: Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors

Abstract Background: Hyper-activation of the PI3K pathway due to a functional loss of PTEN is a characteristic feature of many tumors including TNBC and GBM. Although p110b rarely undergoes activating mutations in these cancers, loss of PTEN function makes these tumor cells dependent specifically on p110b. In a mechanism-based study we have recently established an integral role of the PI3K pathway in DNA damage repair (DDR)-mediated anti-tumor efficacy of PARP inhibitor wherein we have demonstrated that PTEN-nullness confers in vivo sensitivity to the combination as compared to tumors carrying PTEN WT (De et al., Neoplasia, 2014). Thus here we hypothesize that PTEN-null tumors will be more susceptible to a combination of p110b inhibitor (p110b i) plus PARP inhibitor (PARP i). Method: To test our hypothesis we used PTEN-null TNBC and GBM models. Isogenic cell lines as well as SiRNA-directed PTEN-knockdown cells were tested for the (a) IC50 (MTT) (b) real-time confluence assay (Incucyte) (c) cell cycle (flow cytometry) (d) clonogenecity (ON-TOP assay) (e) cytosolic proliferation and apoptotic signals and (f) nuclear HRD signals for DDR using 3 p110b i (TGX221, AZD6482, GSK2636771) and 5 PARPi (BMN673, Niraparib, Olaparib, Rucaparib, Veliparib) (procured from Selleckchem, USA). Results: Individually (1) AZD6482 was the most effective among p110b i in controlling the cell cycle when tested in PTEN null U87MG GBM cells and (2) AZD6482 had the lowest dose (1-10 uM) for achieving the minimum effective dose (MED) in the confluence studies in TNBC and GBM models as compared to other p110b i. The MED of 5 PARPi for TNBC and GBM cells were determined by clonogenic assay (7-10 days). The most potent among 5 PARPi was BMN673 with a dose range of 100 nM-500 nM. Based on the effectiveness of individual drugs, we used a combination dose range of 500 nM-10 uM of 5 PARPi with 10 uM fixed dose of p110b i, AZD6482, and tested the effect of this combination using (1) real time apoptosis assay (Incucyte) and (2) 3D clonogenic assay. The combination of each PARPi with AZD6482 was more effective in perturbing clonogenic growth of PTEN-null TNBC and GBM cells as compared to the individual agent(s) which can be corroborated with the increased apoptosis in the cells treated with the combination and their effect on cell cycle. Significance: Better understanding of the mechanistic involvement of the combination of p110b i and PARPi in BRCA-competent PTEN null tumors may provide a novel target for future therapeutic intervention and provide insight to PTEN-mediated carcinogenesis in these tumors which eventually will extend the realm of PARP i use in cancer therapy. Citation Format: Jennifer H. Carlson, Yuliang Sun, Xiaoqian Lin, Pradip De, Brian Leyland-Jones, Nandini Dey. Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2015-2561

Abstract 1231: In vitro anticancer activity of imidazo-acridinone (HKH40A) in panels of human patient-derived tumor xenografts (PDXs) and human leukemia and lymphoma cell lines

Abstract HKH40A is a synthetic, DNA-intercalating (Kosakowska-Cholody et al., 2009) anticancer agent consisting of an imidazo-acridinone linked to a 1,8-naphthalimide moiety via a 1,4-dipropylpiperazine linker. It displayed strong inhibitory activity in the NCI-60 tumor cell line panel in vitro with an IC50 of about 1 nM in selected tumor cell lines (Hariprakasha et al. 2007). In addition, anticancer activity was observed in hepatoma models both in vitro and in vivo. We have investigated the in vitro anticancer activity of HKH40A in 3D cultures, using panels of 206 PDXs and 11 human leukemia and lymphoma cell lines. The 3D clonogenic assay was performed in a 96 well format using a metabolic readout as an equivalent for colony formation. PDXs and hematologic cell lines were propagated in immunedeficient mice. HKH40A proved to be very potent with a mean IC50 of 6 nM and a mean IC70 of 12 nM. Overall, the hematological cell lines were more sensitive than the solid tumor models. Selective activity (definition: IC50 < 1/3 of mean IC50) was seen in 4 out of 6 acute myeloid leukaemias (AMLs), in 1 out of 1 chronic lymphocytic leukemia (CLL) and in 1 out of 3 non-Hodgkin lymphomas (NHLs). Among the solid tumors, pleural mesotheliomas were most sensitive followed by soft tissue sarcomas and anal cancer. Selective activity was observed in a subset of 8 out of 30 colon cancer models, which presented IC50 values < 1/3 of the mean IC50 of all tumors. The drug sensitivity data will be correlated to mutational and transcriptomic (Whole Exome Sequencing and Affymetrix HG-U133 plus 2.0 array profiles) characteristics of the PDXs to identify predictive biomarkers of drug response and resistance. Overall, HKH40A is a novel imidazo-acridinone with high in vitro activity mainly in AMLs and NHLs as well as in pleural mesotheliomas, soft tissue sarcomas and in a subset of colon cancer models. The identification of predictive biomarkers is ongoing. *Deceased during an NCI retreat Citation Format: Heinz H. Fiebig, Armin Maier, Gerhard Kelter, Vincent Vuaroqueaux, Humcha K. Hariprakasha, Christopher Michejda. In vitro anticancer activity of imidazo-acridinone (HKH40A) in panels of human patient-derived tumor xenografts (PDXs) and human leukemia and lymphoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2014-1231

The Expression of CD90/Thy-1 in Hepatocellular Carcinoma: An In Vivo and In Vitro Study

Although the CD90 (Thy-1) was proposed as biomarker of several tumors and cancer stem cells, the involvement of this molecule in the progression of hepatocellular carcinoma (HCC) and other less frequent hepatic neoplasms is still undefined. The distribution of CD90 was investigated both in in vivo (human tissues samples) and in vitro (human HCC cell line JHH-6). A total of 67 liver tumors were analyzed: 51 HCC, 6 cholangiocarcinoma and 10 hepatoblastoma. In all cases, paired tissue sample of both the tumor and cirrhotic liver was available. Hepatic tissue obtained in 12 healthy livers was used as control. CD90 gene expression was studied by RT-qPCR, protein expression was assessed by quantitative Western Blot, immunofluorescence and flow cytometry. The CD90 expression analysis showed a significant increment in tumor compared to both its paired cirrhotic tissue and normal liver (p<0.05 and p<0.001, respectively). This increase was accompanied by the up-regulation of stromal component in the cancer, as demonstrated by alpha smooth muscle actin staining. In vitro analysis of JHH-6 cell line showed a higher proliferation capacity of CD90+ compared to CD90- cells (p<0.001), also noticed in 3D clonogenic assay (p<0.05), associated by a significant higher expression of the promoting factors (hepatocyte growth factor, fibroblast associated protein and alpha smooth muscle actin 2). A higher expression of the breast cancer resistance protein was found in CD90+ subpopulation while the multidrug resistance protein 1 showed an opposite behavior. Collectively, these results point to the importance of CD90 in the HCC.

Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells

Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.

Abstract PD02-07: Impact of Mesenchymal Stem Cells on Radiation Therapy Response

Abstract Background: Radiation increases engraftment of human bone-marrow derived mesenchymal stem cells (MSC) in tumors and this effect is augmented following repeated doses of radiation. We have recently demonstrated that MSC increase formation of mammospheres, which are enriched with radiation resistant tumor-initiating cells. Thus, we hypothesized that MSC may impact radiation therapy response through enhanced survival of tumor-initiating cells. Materials &amp; Methods: To evaluate the impact of MSC on radiation response we performed in vitro clonogenic survival assays using 2D and 3D clonogenic assays. Colony (2D) and sphere (3D) formation was quantified following irradiation of different cell lines (breast, lung, head and neck and cervix) at 2, 4 and 6 Gy in the presence of MSC conditioned media. In vivo, MSC were co-injected subcutaneously at increasing percentages with 4T1 murine breast cancer cells in BALB/c mice. The effects of MSC on radiation response were evaluated by examination of tumor size and weight following tumor irradiation at 2 and 5 Gy. Results: In vitro 2D clonogenic cell survival assays demonstrated that MSC conditioned media increased survival of breast (MDA231), lung (H460), head and neck (HN5), and cervical (SiHa) cancer cells following irradiation with 2 and 4 Gy as compared to control fibroblast conditioned media. Moreover, MSC conditioned media increased the number of HMLE (breast normal ephitelial cells) and SUM149 (breast cancer cells) mammospheres formed (55 vs 195and 263 vs 1054, respectively) and increased clonogenic survival of mammospheres after 2, 4 and 6 Gy compared to serum-free media. In vivo, irradiation with 5 Gy reduced murine breast 4T1tumors weight by 75% as compared to control untreated tumors. However, tumors co-injected with MSC did not significantly respond to 5 Gy. Discussion: MSC increase tumor resistance to radiation therapy in vivo and in vitro in 2D and 3D cultures. This effect is mediated at least in part by paracrine factors secreted by MSC. Our results here described suggest that MSC secrete factors which activate survival pathways on cancer cells exposed to sub-lethal radiation doses. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-07.