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A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy.

Lang DS, Droemann D, Schultz H, Branscheid D, Martin C, Ressmeyer AR, Zabel P, Vollmer E, Goldmann T - Respir. Res. (2007)

Bottom Line: Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously.Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany. dlang@fz-borstel.de

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC.

Methods: In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines.

Results: Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.

Conclusion: Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.

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Caspase-3 induction determined by flow cytometry in human cancer cell lines in the absence (RPMI) or presence of carboplatin (CARB), vinorelbine (VIN) and gemcitabine (GEM), respectively. The cell lines were plated 24 h before addition of the single drug. Percentage of caspase-3 positive cells are shown as mean values + SEM (n = 4–5).
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Figure 2: Caspase-3 induction determined by flow cytometry in human cancer cell lines in the absence (RPMI) or presence of carboplatin (CARB), vinorelbine (VIN) and gemcitabine (GEM), respectively. The cell lines were plated 24 h before addition of the single drug. Percentage of caspase-3 positive cells are shown as mean values + SEM (n = 4–5).

Mentions: Caspase-3 induction, as determined by flow cytometry, was highly variable in response to carboplatin and gemcitabine (Fig. 2). Vinorelbine appeared to enhance caspase-3 expression more effectively than the two other drugs. Unfortunately, CPC-N cells showed drastically reduced viability to less than 20–30% upon resuspension, which is why data regarding caspase-3 expression had to be acquired by IHC. Here, CPC-N cells demonstrated negligible caspase-3 expression without treatment, but revealed consistent induction up to 30% in response to each cytotoxic drug (Figs. 3A–D).


A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy.

Lang DS, Droemann D, Schultz H, Branscheid D, Martin C, Ressmeyer AR, Zabel P, Vollmer E, Goldmann T - Respir. Res. (2007)

Caspase-3 induction determined by flow cytometry in human cancer cell lines in the absence (RPMI) or presence of carboplatin (CARB), vinorelbine (VIN) and gemcitabine (GEM), respectively. The cell lines were plated 24 h before addition of the single drug. Percentage of caspase-3 positive cells are shown as mean values + SEM (n = 4–5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1913052&req=5

Figure 2: Caspase-3 induction determined by flow cytometry in human cancer cell lines in the absence (RPMI) or presence of carboplatin (CARB), vinorelbine (VIN) and gemcitabine (GEM), respectively. The cell lines were plated 24 h before addition of the single drug. Percentage of caspase-3 positive cells are shown as mean values + SEM (n = 4–5).
Mentions: Caspase-3 induction, as determined by flow cytometry, was highly variable in response to carboplatin and gemcitabine (Fig. 2). Vinorelbine appeared to enhance caspase-3 expression more effectively than the two other drugs. Unfortunately, CPC-N cells showed drastically reduced viability to less than 20–30% upon resuspension, which is why data regarding caspase-3 expression had to be acquired by IHC. Here, CPC-N cells demonstrated negligible caspase-3 expression without treatment, but revealed consistent induction up to 30% in response to each cytotoxic drug (Figs. 3A–D).

Bottom Line: Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously.Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany. dlang@fz-borstel.de

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC.

Methods: In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines.

Results: Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.

Conclusion: Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.

Show MeSH
Related in: MedlinePlus