Tumor Clonogenic Assay Research Articles

306 (PB086) - Reversal of resistance to docetaxel and cabazitaxel in prostate cancer cells with ritonavir

Background: The taxanes docetaxel and cabazitaxel are indicated for treatment of metastatic castration-resistant prostate cancer (mCRPC). Resistance to docetaxel often develops during treatment, which can be due to an increased efflux of docetaxel via P-glycoprotein (P-gp) from the intracellular site of action. Ritonavir is an inhibitor of both cytochrome P4503A4 (CYP3A4) and P-gp. In this study we investigated whether ritonavir could improve the efficacy of docetaxel through inhibition of P-gp in docetaxel-resistant prostate cancer cells. Methods: Prostate cancer cell lines DU-145 and 22Rv1 were exposed to increasing doses of docetaxel to generate resistant sublines, DU145-DOC10 and 22Rv1DOC8 respectively. Gene expression of the resistant clones was compared to that of the parental cell lines. The parental cell lines and the docetaxel resistant cell lines were treated with several drug combinations in an 8*2 matrix 3D tumor clonogenic assay. The following drug combinations were tested: docetaxel/ritonavir, docetaxel/elacridar and cabazitaxel/ritonavir. The IC50 values of single agent drugs and drug combinations as well as combination effects between drugs (using a Bliss independence dose-response evaluation) were determined in all cell lines. Results: The most differentially upregulated gene in the resistant cell lines was ABCB1 (P-gp). Western blotting showed that both resistant cell lines expressed more P-gp than the parental cell lines. A comparison of the observed docetaxel and cabazitaxel IC50 values has been summarized in the below table.Table(abstract: 306 (PB086))DU-145DU-145DOC10DU-145DOC1022Rv122Rv1DOC822Rv1DOC8Docetaxel IC50 (nM)2.99.52.8*with 10 μM ritonavir,1.354.63.0**with 32 μM ritonavir.Cabazitaxel IC50 (nM)0.61.50.6*with 10 μM ritonavir,0.79.50.7**with 32 μM ritonavir.* with 10 μM ritonavir,** with 32 μM ritonavir. Open table in a new tab We found that docetaxel resistance conferred cross-resistance to cabazitaxel. With the addition of 10 μM ritonavir (DU-145DOC10) and 32 μM ritonavir (22Rv1DOC8) the cytoxicity of docetaxel and cabazitaxel returned to the level of observed in the parental cell lines. A similar experiment with the specific P-gp inhibitor elacridar showed that a concentration of 0.3 μM was sufficient to reverse docetaxel resistance to the same level as in the parental cell lines, confirming this as an important mechanism-of-action. In DU-145-DOC10 and 22Rv1DOC8 cells strong synergistic effects were seen of the docetaxel/ritonavir as well as the cabazitaxel/ritonavir combination, in contrast to the lack of synergy seen in the parental cell lines with these combinations. Conclusions: We demonstrated the potential for the combination of docetaxel and ritonavir to reverse or offset resistance to docetaxel (and, by implication due to its cross-resistance, also to cabazitaxel), primarily mediated through inhibition of P-gp. This provides a strong rationale to clinically test these taxanes in combination with ritonavir in patients with mCRPC. Conflict of interest: Ownership: Jos Beijnen is a shareholder of Modra Pharmaceuticals. Board of Directors: Colin Freund and Eric van der Putten are directors of Modra Pharmaceuticals.

Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect

While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.Graphical abstract

Inter/intra-tumoral dose response variations assessed using FDG-PET/CT feedback images: Impact on tumor control and treatment dose prescription

PurposeTo quantify inter/intra-tumoral variations of baseline metabolic activity and dose response. To evaluate their impact on tumor control and treatment dose prescription strategies. Methods and materialsTumor voxel baseline metabolic activity, SUV0, and dose response matrix, DRM, quantified using the pre-treatment and weekly FDG-PET/CT imaging feedback for each of 34 HNSCC patients (25 HPV+ and 9 HVP−) were evaluated. Inter/intra-tumoral variations of tumor voxel (SUV0, DRM) for each of the HPV− and HPV+ tumor groups were quantified and used to evaluate the variations of individual tumor control probabilities and the efficiency of uniform vs non-uniform treatment dose prescription strategies. ResultsTumor voxel dose response variation of all tumor voxels assessed using FDG-PET/CT imaging feedback had the mean(CV) = 0.47(47%), which was consistent with those of previously published in vitro tumor clonogenic assay. The HPV− tumors had the mean(CV) dose response, 0.53(49%), significantly larger than those of the HPV+ tumors, 0.45(43%). However, their baseline SUVs were opposite, 6.5(56%) vs 7.7(65%). Comparing to the inter-tumoral variations, both HPV−/+ tumor groups showed larger intra-tumoral variations, (53%, 58%) vs (20%, 31%) for the baseline SUV and (38%, 37%) vs (31%, 21%) for the dose response. Due to the large dose response variations, treatment dose to control the tumor voxels has very broad range with CV of TCD50 = 97% for the HPV− and 67% for the HPV+ tumor group respectively. As a consequence, heterogeneous prescription dose could potentially reduce the treatment integral dose for 92% of the HPV+ tumors and 78% of the HPV− tumors. ConclusionsThe study demonstrates that tumor dose response assessed using FDG-PET/CT feedback images had a similar distribution to those assessed conventionally using in vitro tumor clonogenic assay. Inter-tumoral dose response variation seems larger for HPV− tumors, but intra-tumoral dose response variations are similar for both HPV groups. These variations cause very large variation on the individual tumor control probability and limit the efficacy of dose escalation and de-escalation in conventional clinical practice. On the other hand, heterogeneous dose prescription guided by metabolic imaging feedback has a potential advantage in radiotherapy.

The effect of CELLFOODTM on radiotherapy or combined chemoradiotherapy: preclinical evidence.

Background:Based on previous observations that the nutraceutical CELLFOOD™ (CF), the ‘physiological modulator’ that aimed to make oxygen available ‘on demand’, inhibits the growth of cancer cells, this study was designed to investigate the role of CF in the regulation of hypoxia-inducible factor 1 alpha (HIF1α) and its correlated proteins, phosphoglycerate kinase 1 and vascular endothelial growth factor. Our idea was that CF, acting on HIF1α, in combination with current anticancer therapies could improve their effectiveness.Methods:To evaluate the effect of CF in association with radiotherapy and chemotherapy, different human cancer cell lines and mice with mesothelioma were analysed by tumour growth, clonogenic assay, western blot and immunohistochemical analysis.Results:CF in combination with radiation with or without cisplatin increases the death rate of cancer cells. In vivo, 70% of mice treated with CF before the mesothelioma graft did not show any tumour growth, indicating a possible preventive effect of CF. Moreover, in mouse mesothelioma xenografts, CF improves the effect of radiotherapy also in combination with chemotherapy treatment. Immunohistochemical analysis of tumour explants showed that HIF1α expression was reduced by the combination of CF and radiotherapy treatment and even more by the combination of CF and radiotherapy and chemotherapy treatment. Mechanistically, CF increases the fraction of oxygenated cells, making the radiotherapy more effective with a greater production of reactive oxygen species (ROS) that in turn, reduce the HIF1α expression. This effect is amplified by further increase in ROS from chemotherapy.Conclusions:Collectively, results from preclinical trials suggest that CF could be a useful intervention to improve the efficacy of radiotherapy or combined treatment strategies and could be a promising treatment modality to counteract cancer.

Abstract 4835: UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers

Abstract Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer activities of UD-017 in vitro and in vivo using patient-derived (PD) cancer cells. Methods: We first evaluated an antiproliferative activity of UD-017 broadly in a panel of cancer cell assay including patient-derived 3-dimensional tumor clonogenic assay. To verify the potential of c-Myc expression as a biomarker, we investigated the correlation between c-Myc expression levels and anticancer activity in vitro and in vivo using a colorectal cancer cell. In the main part, anticancer efficacy of UD-017 was investigated in vivo in PD-xenograft (PDX) models using representative PD cancer cells. Results: In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas, osteosarcoma, sarcoma and urinary bladder cancers. Especially, UD-017 showed high sensitivity in solid tumor such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, osteosarcoma and sarcoma cells with IC50s of 10-200 nM. UD-017 showed a good correlation of c-Myc expression levels with antiproliferative activity using colorectal cancer cells in vitro, and UD-017 reduced the intratumoral MYC mRNA levels by an administration at 100 mg/kg in a HCT-116 xenograft model in mice. In PDX models, UD-017 showed strong inhibition and even regression of tumor growth, and the tumors were almost disappeared on day 14 without body weight loss in non-small cell lung cancer (LXFL1121). In a pleuramesothelioma (PXF541) PDX model, UD-017 also showed regressive effect, and in gastric cancer (GXA3067) and sarcoma (SXFS117) PDX models, UD-017 completely inhibited the tumor growth. Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete antitumor responses in patient-derived xenograft models of diverse cancer types. c-Myc expression in cancer cells may be a biomarker for the antitumor effect of UD-017. These data support the rationale for further advancing towards clinical development. Citation Format: Takashi Matsushita, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Toru Hasegawa, Yasunori Tokunaga, Yasuhiro Aga, Shigeru Ushiyama. UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4835.

Abstract 3699: Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts

Abstract Patients with metastatic V600E mutant melanomas treated with BRAF inhibitors (BRAFi) frequently develop resistant tumors with aggressive phenotypes. Furthermore, approximately 20-40% of V600E mutant tumors are intrinsically resistant to BRAFi. Suitable models for studying mechanisms of acquired and intrinsic resistance to BRAFi are therefore necessary. In the present study, we applied deep sequencing techniques to identify possible mechanisms of intrinsic and acquired resistance in our collection of melanoma patient-derived xenograft models (MEXFs) treated with BRAFi. Mutational and expression profiles in MEXFs were characterized by whole-exome sequencing (WES) and HG-U133 Plus 2.0 Affymetrix chips and correlated with BRAFi efficacy data from 3D Tumor Clonogenic Assays (TCA). In addition, four resistant cell lines were created by continuously treating 2D monolayer cultures of tumor cells with Vemurafenib, all of which were initially sensitive to BRAFi and carry the BRAF V600E mutation. Expression profiles and mutations of these cell lines were analyzed from RNA-seq data. Potential gene candidates responsible for conferring resistance were further investigated by Q-PCR and Western-blot experiments. WES data revealed that the number of mutations per model was highly variable. Some MEXFs showed a hyper-mutated profile (&amp;gt;2000 mutations) and were characterized by specific mutational signatures in agreement with those found in melanoma (Alexandrov et al, Nature, 2013). Mutation frequencies of genes typically mutated in melanoma are very similar to those found in The Cancer Genome Atlas. We observed a high correlation of mutations between our MEXFs and their respective cell lines. Among the models with a V600E mutation, only one (MEXF 462) showed resistance to BRAFi-treatment as identified by 3D TCA analyses. In this model, we identified gene point mutations and a high over-expression of EGFR, MEK1, PDGFRA and NF1 in contrast to the other models, suggesting a specific regulation of the RTK signaling pathways. In 2D assays, synergistic interaction of Vemurafenib and Erlotinib was shown. RNA-seq analysis of the cell line established from MEXF 276, in which resistance to Vemurafenib was induced, revealed around 20% of genes being differentially expressed (FC&amp;gt;2) between sensitive and resistant cell lines. An up-regulation of EGFR was also found in this resistant cell line and was confirmed by Western-blot. In addition, an overexpression of PLAU, a biomarker of invasiveness, was identified. We currently perform expression profiling of three other models resistant to BRAFi. Preliminary results suggest different patterns of gene regulation involved in acquisition of resistance. A precise map of transcriptomic and mutational profiles of the four cell lines will be generated and we are investigating if invasiveness is increased upon acquisition of resistance. Citation Format: Bruno Zeitouni, Gerhard Kelter, Armin Maier, Florian Kiefer, Frederic Foucault, Anne-Lise Peille, Tim Kees, Torsten Giesemann, Vincent Vuaroqueaux, Thomas Metcalfe, Heinz-Herbert Fiebig. Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts. [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 3699. doi:10.1158/1538-7445.AM2014-3699

Sunitinib effects on the radiation response of endothelial and breast tumor cells

BackgroundEndothelial cells are suggested regulators of tumor response to radiation. Anti-vascular targeting agents can enhance tumor response by targeting endothelial cells. Here, we have conducted experiments in vitro to discern the effects of radiation combined with the anti-angiogenic Sunitinib on endothelial (HUVEC) and tumor (MDA-MB-231) cells, and further compared findings to results obtained in vivo. MethodsIn vitro and in vivo treatments consisted of single dose radiation therapy of 2, 4, 8 or 16Gy administered alone or in combination with bFGF or Sunitinib. In vitro, in situ end labeling (ISEL) was used to assess 24-hour apoptotic cell death, and clonogenic assays were used to assess long-term response. In vivo MDA-MB-231 tumors were grown in CB-17 SCID mice. The vascular marker CD31 was used to assess 24-hour acute response while tumor clonogenic assays were used to assess long-term tumor cell viability following treatments. ResultsUsing in vitro studies, we observed an enhanced endothelial cell response to radiation doses of 8 and 16Gy when compared to tumor cells. Administering Sunitinib alone significantly increased HUVEC cell death, while having modest additive effects when combined with radiation. Sunitinib also increased tumor cell death when combined with 8 and 16Gy radiation doses. In comparison, we found that the clonogenic response of in vivo treated tumor cells more closely resembled that of in vitro treated endothelial cells than in vitro treated tumor cells. ConclusionOur results indicate that the endothelium is an important regulator of tumor response to radiotherapy, and that Sunitinib can enhance tumor radiosensitivity. To the best of our knowledge, this is the first time that Sunitinib is investigated in combination with radiotherapy on the MDA-MB-231 breast cancer cell line.

Abstract C30: A KRAS pathway activation index predicting response to MEK inhibitors in patient-derived tumor xenografts.

Abstract Introduction: MEK 1/2 inhibitors (MEKi) are promising compounds for the treatment of cancer due to frequent activation of the RAS/MAPK/ERK oncogenic pathway. CI-1040 and PD0325901 are newly developed MEKi that are currently being tested in clinical trials. In the present study, we investigated MEKi response in different tumor types and we determined whether an index of KRAS pathway activation (K-PAI) could predict response to MEKi. Material and Methods: CI-1040 and PD0325901 were tested using an ex vivo 3D Tumor Clonogenic Assay (TCA) in a panel of 63 patient-derived tumor xenografts (PDX) covering 15 tumor histotypes. The K-PAI was determined by identifying gene expression patterns (Affymetrix HGU133 plus 2.0 arrays) associated with activation of the pathway and KRAS mutational status (determined by Sanger sequencing). Results: The absolute activities (IC50) of CI-1040 and PD0325901 correlated in most of the tumor models tested (r=0.87). Most of the melanomas were sensitive to both MEKi tested, whereas variable response profiles were observed in colon cancers and non-small cell lung cancers (NSCLC). Ovarian and pancreatic cancer xenografts displayed in most instances weak responses. The KRAS and BRAF statuses were significantly associated with MEKi IC50 (p=0.0001 and p=0.0002, respectively). The melanomas which frequently displayed BRAF mutations (13/21), were highly sensitive to MEKi treatment, whereas ovarian and pancreatic tumors, which frequently harbored KRAS mutations (1/3 and 2/2), were resistant. Moreover, we found that the K-PAI correlated significantly with MEKi IC50 (r&amp;gt;0.5, p&amp;lt;0.0001 for CI-1040 and PD0325901).= Melanomas with low K-PAI values were highly sensitive to MEKi treatment whereas ovarian and pancreatic tumors with high K-PAI values were resistant. Interestingly, the K-PAI was also predictive of response to MEKi treatment for tumors expressing wild-type BRAF and KRAS.Conclusion: This large ex vivo PDX study showed that tumor sensitivity to MEKi is related to histology and to RAS pathway activation. KRAS and BRAF mutations were predictive of MEKi response (resistance and sensitivity, respectively). These results are consistent with published data on cell lines and on small patient cohorts and demonstrate that PDX are adequate models to test targeted drugs such as MEKi. The correlation of tumor sensitivity to MEKi and the RAS pathway activation level (K-PAI) should be further evaluated in ex vivo 3D assays utilizing PDXs or with an in vivo study, to be able to better stratify patients when testing the predictive potential of K-PAI in clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C30. Citation Format: Anne-Lise Peille, Armin Maier, Frederic Foucault, Rebekka Krumbach, Tim Kees, Torsten Giesemann, Thomas Metz, Thomas Metcalfe, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. A KRAS pathway activation index predicting response to MEK inhibitors in patient-derived tumor xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C30.

Abstract 3835: Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors.

Abstract For compounds directed against molecular targets expressed in tumor cells, the ex vivo 3D tumor clonogenic assay (TCA) is a rapid and reliable ex vivo assay with a high predictive value for in vivo tumor sensitivity. Single cell suspensions prepared from patient-derived tumor xenografts (PDX) growing subcutaneously in nude mice or from cultured human tumor cell lines are seeded in semisolid medium and tumor colony formation is monitored in the presence or absence of test compounds over a period of one to three weeks. Based on experiments with up to 70 PDX, we have demonstrated that the TCA accurately replicates the in vivo sensitivity of PDX towards cMet inhibitors across all major tumor histologies. More specifically, all three NSCLC PDX that regressed in response to cMet inhibition in in vivo efficacy tests were sensitive to several cMet inhibitors in the ex vivo TCA. By contrast, data obtained with a 2D cell proliferation and survival assay did not correlate with the 3D and in vivo situations, suggesting that cMet function does not affect cell survival and proliferation on plastic but confers the capacity for anchorage-independent growth. The correlation of 3D but not 2D data with in vivo sensitivity was confirmed using anti-cMet siRNAs in selected PDX-derived non-small cell lung cancer cell lines. Preliminary immunohistochemical analysis revealed that PDX sensitive to cMet inhibitors expressed high cMet levels while not all PDX expressing high cMet levels were sensitive to cMet inhibitors. In conclusion, for cMet inhibitors the TCA replicates in vivo sensitivities of PDX to a high degree. Due to its short duration the TCA is an excellent tool for the screening of large numbers of cMet inhibitors and PDX. As all of the PDX qualified for use in the TCA (&amp;gt;200) have been extensively molecularly characterised (gene expression, gene copy number variation and mutation analysis) this assay is also an excellent tool for generating high quality biomarker hypotheses during the preclinical profiling of molecules intended for oncology indications. Citation Format: Sabine Gorynia, Jianing Guo, Andreas Ackermann, Armin Maier, Rebekka Krumbach, Gerhard Kelter, Vincent Vuaroqueaux, Thomas Metz, Thomas Metcalfe, Heiner H. Fiebig. Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2013-3835

In vitro stemness characterization of radio-resistant clones isolated from a medulloblastoma cell line ONS-76

One-third of patients with medulloblastoma die due to recurrence after various treatments including radiotherapy. Although it has been postulated that cancer stem-like cells are radio-resistant and play an important role in tumor recurrence, the “stemness” of medulloblastoma cells surviving irradiation has not yet been elucidated. Using a medulloblastoma cell line ONS-76, cells that survived gamma irradiation were investigated on their “stemness” in vitro. From 10 500 cells, 20 radio-resistant clones were selected after gamma ray irradiation (5 Gy × two fractions) using the replica micro-well technique. These 20 resistant clones were screened for CD133 positivity by flow cytometry followed by side population assay, tumor sphere formation assay and clonogenic survival assay. Results revealed CD133 fractions were significantly elevated in three clones, which also exhibited significantly increased levels of tumor sphere formation ability and side population fraction. Clonogenic survival assay demonstrated that their radio-resistance was significantly higher than the parental ONS-76. This may support the hypothesis that a small number of cancer stem-like cells (CSCs) are the main culprits in local recurrence after radiotherapy, and disruption of the resistance mechanism of these CSCs is a critical future issue in improving the outcome of patients with medulloblastoma.

Activated Marrow-Infiltrating Lymphocytes Effectively Target Plasma Cells and Their Clonogenic Precursors

A major limitation of adoptive immunotherapy is the availability of T cells specific for both terminally differentiated tumor cells and their clonogenic precursors. We show here that marrow-infiltrating lymphocytes (MILs) recognize myeloma cells after activation with anti-CD3/CD28 beads with higher frequency than activated peripheral blood lymphocytes from the same patients. Furthermore, activated MILs target both the terminally differentiated CD138+ plasma cells and the myeloma precursor as shown by profound inhibition in a tumor clonogenic assay. The presence of antigen in the marrow microenvironment seems to be important for the maintenance of tumor specificity. Taken together, these results highlight the intrinsic tumor specificity of MILs and describe a novel approach for the generation of tumor-specific T-cell populations suitable for adoptive immunotherapy of multiple myeloma.

Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

ATP chemosensitivity testing in ovarian and breast cancer: early clinical trials.

After disappointing results achieved with older chemosensitivity tests such as the human tumor clonogenic assay (HTCA) during the 1980s, the last decade has seen a renaissance of the concept of individualized chemotherapy in oncology, markedly stimulated by the development of newer nonclonogenic assays. These methods appear to be able to overcome major technical limitations associated with older assays, now allowing for successful testing of most of the tumor specimens submitted. Currently, the ATP-based tumor chemosensitivity assay (ATP-TCA) can be regarded as the most sophisticated assay to investigate both solid samples and effusions derived from patients with various organ tumors. During the last 5 years, the ATP-TCA has been used successfully to screen for novel drug combinations for further clinical use in both ovarian and breast cancer such as mitoxantrone plus paclitaxel (NT) and treosulfan plus gemcitabine (TG), respectively. Clinical trials that have been set up in heavily pretreated patients with recurrent ovarian or breast cancer have convincingly confirmed the high activity of these combinations previously demonstrated in preclinical investigations using the ATP-TCA. In a recent phase II trial performed in 59 patients with relapsed ovarian carcinoma, ATP-TCA-directed therapy was able to triple the response rate and to double the survival time, compared with published empirical chemotherapy regimes. Preliminary results with ATP-TCA-directed therapy in breast cancer also evidenced promising response rates. These results have been confirmed by additional prospective clinical trials using other types of modern nonclonogenic assays. A phase III trial that is now actively recruiting patients with platinum-refractory ovarian cancer to verify the promising phase II studies will prove the further value of the ATP-TCA as a predictor applicable in routine clinical oncology.

Induction of retinoic acid receptor β mediates growth inhibition in retinoid resistant human colon carcinoma cells

BACKGROUNDThe molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood.AIMTo identify the intracellular mechanisms responsible for resistance against retinoid mediated...

Retinoic acid receptor γ 1 expression determines retinoid sensitivity in pancreatic carcinoma cells

Background & Aims: Retinoids inhibit growth and induce differentiation in a variety of pancreatic carcinoma cells. The goal of this study was to examine the molecular mechanisms responsible for retinoid sensitivity. Methods: Anchorage-independent growth was examined in AR42J, DSL-6A/C1, and Capan-2 cells using a human tumor clonogenic assay. Retinoid receptors were characterized by a reverse-transcription polymerase chain reaction. Retinoic acid receptor γ 1 (RARγ 1) was stably transfected into AR42J cells using lipofectamin and into DSL-6A/C1 using ballistomagnetic gene transfer. Receptor expression was verified using Southern and Northern blotting as well as electrophoretic mobility shift assays. Results: Retinoid treatment resulted in a dose-dependent growth inhibition of Capan-2 cells, whereas growth was not affected in AR42J and DSL-6A/C1 cells. A selective loss of RARγ 1 expression was observed in both retinoid-resistant cell lines, whereas all other retinoid receptor subtypes showed an identical expression pattern. Retinoid treatment of three independent RARγ 1-expressing cell clones of AR42J and DSL-6A/C1 cells resulted in pronounced growth inhibition compared with wild-type control cells. Conclusions: RARγ 1 expression determines sensitivity of pancreatic carcinoma cells to retinoid-mediated growth inhibition and might therefore serve as a valuable predictive marker for retinoid treatment of pancreatic cancer. GASTROENTEROLOGY 1998;115:967-977

Induction of retinoic acid receptor β mediates growth inhibition in retinoid-resistant human colon carcinoma cells

The molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood.To identify the intracellular mechanisms responsible for resistance against retinoid mediated growth inhibition in human colon carcinoma cells.Anchorage independent growth of the human colon carcinoma cell lines HT29 and LoVo was determined by a human tumour clonogenic assay. Retinoid receptor expression was evaluated by reverse transcription polymerase chain reaction and northern blotting. Retinoid mediated transactivation was assessed by transient transfection of a pTK::betaREx2-luc reporter construct. Retinoid receptor overexpression was achieved by selecting stably transfected cell clones.Retinoid treatment resulted in profound dose dependent growth inhibition in HT29 cells, while LoVo cells were unaffected. The two cell lines express identical patterns of nuclear retinoid receptor mRNA transcripts. However, on retinoid treatment, retinoic acid receptor beta gene expression was upregulated only in retinoid sensitive HT29 cells, but not in retinoid resistant LoVo cells. In accordance, stable overexpression of retinoic acid receptor beta but not alpha or gamma conferred retinoid mediated growth inhibition on LoVo cells.Induction of retinoic acid receptor beta expression is required and sufficent to confer retinoid mediated growth inhibition on human colon carcinoma cells.

頭頚部癌の抗癌剤耐性とその克服

The incidence of natural chemoresistance in head and neck cancers was 72 to 79% (76% in average), based on estimates obtained from 3 kinds of chemosensitivity tests including the human tumor clonogenic assay, the rapid thymidine incorporation assay, and the ATP assay. The average incidence of sensitivity to anticancer drugs, 24%, was almost the same as the previously reported average efficacy of single drugs, 27%. Several mechanisms of resistance were discussed and some effective compounds to overcome anticancer resistance were reviewed. One established mechanism of multidrug resistance is elevated expression of P-glycoprotein (Pgp). The expression of Pgp was studied using immunohistochemical techniques in 31 squamous cell carcinomas (SCC) using two monoclonal antibodies, C219 and JSB1. Pgp was detected in 61% of the samples. In the CDDP resistant cell line, IMC-3/CR, both a decreased influx and increased efflux of CDDP were observed. Cellular glutathione (GSH) level is thought to be another factor in anticancer drug resistance. Therefore, GSH content in 28 malignant head and neck tumors was measured by high-performance liquid chromatography. SCCs contained significantly higher levels of GSH than thyroid papillary carcinomas. Chemosensitivity to CDDP was negatively correlated with cellular GSH content. Some compounds effective in overcoming drug resistance, such as calcium channel blockers, calmodulin inhibitors, cyclosporin derivatives, amphotericin B, DHA, BSO, caffeine, and pentoxifylline were discussed.

Similar breast cancer cell contamination of single-day peripheral-blood progenitor-cell collections obtained after priming with hematopoietic growth factor alone or after cyclophosphamide followed by growth factor.

To evaluate tumor-cell contamination of peripheral-blood progenitor-cell (PBPC) collections obtained after priming with granulocyte colony-stimulating factor (G-CSF). Immunocytochemical (ICC) and tumor clonogenic (TCA) assays were used to analyze tumor-cell contamination of pretreatment peripheral-blood (PB) and bone marrow (BM) samples, and of PBPC collection samples obtained after priming with G-CSF 5 micrograms/kg/d for 5 or 7 days in 38 women with advanced breast cancer undergoing high-dose chemotherapy (HDC). Results were compared with 37 historical control patients who underwent PBPC mobilization with cyclophosphamide (4 g/m2) followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) 5 micrograms/kg/d for 14 days. Before PBPC priming with G-CSF, only one of 37 (3%) PB and four of 36 (11%) BM samples had tumor cells detected by ICC. Tumor-cell contamination of PBPC collections obtained after 5 or 7 days of G-CSF priming was observed in only three of 38 patients (8%). All patients with tumor cells detected in the PBPC collection had stage IV disease. Cells with in vitro clonogenic potential were detected only in the pretreatment BM sample in one patient, and another two patients had ICC- and TCA-positive PBPC samples despite tumor-negative PB and BM before priming. These results are similar to those previously reported for PBPC primed with cyclophosphamide and GM-CSF. In patients with advanced breast cancer responsive to cytotoxic chemotherapy, tumor-cell contamination is not increased in PBPC collected after 5 or 7 days priming with G-CSF and appears similar to that seen when PBPC are primed with cyclophosphamide followed by GM-CSF.

An attempt to generate an antitumor effect in the regional lymph nodes against endometrial cancer cells by inducing antitumor cytokines.

Phytohemagglutinin-stimulated regional lymph node cells obtained from gynecological cancer patients exerted a significant antiproliferative activity against an endometrial cancer cell line, RL95-2 on a human tumor clonogenic assay, and released a high amount of tumor necrosis factor alpha (TNF alpha), and interferons. The activity was thought to be partly due to released TNF alpha, because RL95-2 was highly sensitive to recombinant TNF alpha. However, anti-TNF alpha failed to inhibit the activity, which indicated the probability of some as yet unclarified participation of cytokines. Therefore, the regional lymph nodes might be used as sites of endogenous cytokine therapy in endometrial cancer patients.

Absence of lysosomal cleavage in the cytotoxicity mechanism of an immunoconjugate composed of anti-alpha-fetoprotein monoclonal antibody and vindesine analog.

The effect of lysosomal enzyme inhibition on the cytotoxic activity of an immunoconjugate composed of anti-alpha-fetoprotein monoclonal antibody and vindesine analog (VDS) was studied in vitro using human tumor clonogenic assay (HTCA). Addition of the lysosome enzyme inhibitors, leupeptin and ammonium chloride, to the HTCA system had little influence on the cytotoxicity of this immunoconjugate. In separate experiments, no released VDS was detected by HPLC after incubation with the supernatant of rat liver homogenate without inhibitor. These results show that the immunoconjugate may bypass the lysosomal process and exert its activity as an intact or similar form.