Analysis of Molecular Events During Lung Cancer Chemotherapy
Analysis of Molecular Events During Lung Cancer Chemotherapy
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.
To date, no effective chemotherapeutic treatment for non-small cell lung cancer (NSCLC) exists. Therefore, this type of tumor is characterized by a poor prognosis with regard to clinically successful chemotherapy and long-term survival of the patients. Little is known about the complex interactions taking place within the human lung upon chemotherapy. One reason for this might be implied by the common models used for analyzing such interactions like cell cultures or animal models, since such experimental data can be transferred only to a limited extent. As part of a large scaled investigation aimed at improving the facilities available today for the treatment of NSCLC, we report the use of an ex vivo tissue culture model (STST: Short-Term Stimulation of Tissues) in combination with the novel HOPE-technique (Hepes Glutamic acid buffer mediated Organic solvent Protection Effect) to gain insight into the cellular events taking place upon conventional chemotherapy.
Such ex vivo models are long known, however; this technique up to date has failed to become widely used in clinical sciences. The major reason for this is due to the fixation of tissues with formalin; although morphology is well maintained, the application of molecular techniques is largely restricted due to degradation of nucleic acids and protein cross-linking. Since drug-induced effects or immunological reactions within the tissue are hardly correlated with morphological but with molecular changes, the application of a better suited fixation technique allowing for molecular read outs would be a step ahead. With the development of the novel HOPE-technique, immunohistochemical detection has been considerably improved and together with excellent preservation of nucleic acids, molecular analyses can be comprehensively applied. The combination of short-term cultivation using vital tissues and HOPE-fixation (STST) has already been described for other functional studies in the human system. To date, there is only one description on the behavior of NSCLC in organ culture, which was based on formalin-fixed, paraffin-embedded specimens and included only a limited number of tissues with no comprehensive molecular read out.
With regard to the high cellular heterogeneity of NSCLC, experimental data are necessary to elucidate the molecular mechanisms underlying tumor behaviour in detail, thus providing the base for the development of individual and more efficient anticancer treatment regimens. However, most experimental data are based on either animal models, the extrapolation of which to humans is limited or on cell lines that cannot mimic both the complexity and heterogeneity of tumor tissues. As a consequence, we hypothesize that the use of vital human lung tumor tissue specimens would provide a promising novel ex vivo model to elucidate the molecular mechanisms underlying tumor behavior in detail, thus providing the base for the development of individual and more efficient anticancer treatment regimens. Furthermore, such a model, in contrast to cell culture, enables to study the influence of inflammatory cells, which can make up a substantial part of the tumor scenario.
In order to evaluate the suitability of this novel short-term ex vivo model (STST) using human NSCLC specimens, we studied possible drug-induced alterations of multiple known relevant biomarkers for human NSCLC. First, the effects of the chosen culture conditions on the viability of tumor tissues were assessed by LIVE/DEAD viability/cytotoxicity assay using 2-photon microscopy in two separate experiments. To analyze the effects of conventional chemotherapy in STST, each of the chemotherapeutic agents including carboplatin, vinorelbine and gemcitabine, was directly applied ex vivo to 41 different human lung tumor specimens of both squamous cell - and adenocarcinoma type for a 16 h culture period. These anticancer drugs are widely used for treatment of NSCLC patients. They prevent cell proliferation by DNA damage or by tubulin disintegration and also inhibit cellular repair mechanisms. Vinorelbine is also known to inactivate bcl-2 by phosphorylation, thus initiating apoptosis.
A series of cell culture experiments using A549 (NSCLC, adenocarcinoma type), CPC-N (SCLC, small-cell lung carcinoma), HeLa, and HEK cell lines was performed under identical chemotherapeutical culture conditions to compare our results with those obtained by an established experimental model regarding both viability and functionality of the cells. Furthermore, specimens of breast cancer were also treated like the lung tumor samples to verify the efficiency of the used drug concentrations on the chosen biomarkers in a chemo-susceptible type of cancer.
After cultivation, tissue samples were fixed using the novel HOPE technique and paraffinized as described elsewhere. After deparaffinization, protein expression of Ki-67 as indication for the proliferative fraction of the tumour cells was assessed by immunohistochemistry. BrdU uptake as a marker for DNA synthesis in activated cells of S phase was also included as test of more functional relevance. An important key regulator of the apoptotic pathway such as caspase-3 was also evaluated immunohistologically to study the induction of apoptosis. For the cell lines, drug-induced expression of caspase-3 was analyzed by flow cytometry. In a limited number of experiments, analyses of specific mRNA of caspase-3 were also performed by reverse transcriptase - polymerase chain reaction (RT-PCR) in order to verify the results obtained on the protein level. In addition, we exemplarily assessed drug-induced DNA strand breaks in apoptotic cells by the TUNEL labelling assay, to further validate the importance of cleaved caspase-3 as a relevant biomarker for apoptosis.
In an ideal setup these data would have been correlated to actual patient responses to treatment; however, not all of the patients subjected to lung surgery will receive chemotherapy. Moreover, these chemotherapeutic interventions usually do not take place in the moment directly after surgery. Nevertheless, such data - if available - will be collected on the long-run.
Abstract and Background
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.
Background
To date, no effective chemotherapeutic treatment for non-small cell lung cancer (NSCLC) exists. Therefore, this type of tumor is characterized by a poor prognosis with regard to clinically successful chemotherapy and long-term survival of the patients. Little is known about the complex interactions taking place within the human lung upon chemotherapy. One reason for this might be implied by the common models used for analyzing such interactions like cell cultures or animal models, since such experimental data can be transferred only to a limited extent. As part of a large scaled investigation aimed at improving the facilities available today for the treatment of NSCLC, we report the use of an ex vivo tissue culture model (STST: Short-Term Stimulation of Tissues) in combination with the novel HOPE-technique (Hepes Glutamic acid buffer mediated Organic solvent Protection Effect) to gain insight into the cellular events taking place upon conventional chemotherapy.
Such ex vivo models are long known, however; this technique up to date has failed to become widely used in clinical sciences. The major reason for this is due to the fixation of tissues with formalin; although morphology is well maintained, the application of molecular techniques is largely restricted due to degradation of nucleic acids and protein cross-linking. Since drug-induced effects or immunological reactions within the tissue are hardly correlated with morphological but with molecular changes, the application of a better suited fixation technique allowing for molecular read outs would be a step ahead. With the development of the novel HOPE-technique, immunohistochemical detection has been considerably improved and together with excellent preservation of nucleic acids, molecular analyses can be comprehensively applied. The combination of short-term cultivation using vital tissues and HOPE-fixation (STST) has already been described for other functional studies in the human system. To date, there is only one description on the behavior of NSCLC in organ culture, which was based on formalin-fixed, paraffin-embedded specimens and included only a limited number of tissues with no comprehensive molecular read out.
With regard to the high cellular heterogeneity of NSCLC, experimental data are necessary to elucidate the molecular mechanisms underlying tumor behaviour in detail, thus providing the base for the development of individual and more efficient anticancer treatment regimens. However, most experimental data are based on either animal models, the extrapolation of which to humans is limited or on cell lines that cannot mimic both the complexity and heterogeneity of tumor tissues. As a consequence, we hypothesize that the use of vital human lung tumor tissue specimens would provide a promising novel ex vivo model to elucidate the molecular mechanisms underlying tumor behavior in detail, thus providing the base for the development of individual and more efficient anticancer treatment regimens. Furthermore, such a model, in contrast to cell culture, enables to study the influence of inflammatory cells, which can make up a substantial part of the tumor scenario.
In order to evaluate the suitability of this novel short-term ex vivo model (STST) using human NSCLC specimens, we studied possible drug-induced alterations of multiple known relevant biomarkers for human NSCLC. First, the effects of the chosen culture conditions on the viability of tumor tissues were assessed by LIVE/DEAD viability/cytotoxicity assay using 2-photon microscopy in two separate experiments. To analyze the effects of conventional chemotherapy in STST, each of the chemotherapeutic agents including carboplatin, vinorelbine and gemcitabine, was directly applied ex vivo to 41 different human lung tumor specimens of both squamous cell - and adenocarcinoma type for a 16 h culture period. These anticancer drugs are widely used for treatment of NSCLC patients. They prevent cell proliferation by DNA damage or by tubulin disintegration and also inhibit cellular repair mechanisms. Vinorelbine is also known to inactivate bcl-2 by phosphorylation, thus initiating apoptosis.
A series of cell culture experiments using A549 (NSCLC, adenocarcinoma type), CPC-N (SCLC, small-cell lung carcinoma), HeLa, and HEK cell lines was performed under identical chemotherapeutical culture conditions to compare our results with those obtained by an established experimental model regarding both viability and functionality of the cells. Furthermore, specimens of breast cancer were also treated like the lung tumor samples to verify the efficiency of the used drug concentrations on the chosen biomarkers in a chemo-susceptible type of cancer.
After cultivation, tissue samples were fixed using the novel HOPE technique and paraffinized as described elsewhere. After deparaffinization, protein expression of Ki-67 as indication for the proliferative fraction of the tumour cells was assessed by immunohistochemistry. BrdU uptake as a marker for DNA synthesis in activated cells of S phase was also included as test of more functional relevance. An important key regulator of the apoptotic pathway such as caspase-3 was also evaluated immunohistologically to study the induction of apoptosis. For the cell lines, drug-induced expression of caspase-3 was analyzed by flow cytometry. In a limited number of experiments, analyses of specific mRNA of caspase-3 were also performed by reverse transcriptase - polymerase chain reaction (RT-PCR) in order to verify the results obtained on the protein level. In addition, we exemplarily assessed drug-induced DNA strand breaks in apoptotic cells by the TUNEL labelling assay, to further validate the importance of cleaved caspase-3 as a relevant biomarker for apoptosis.
In an ideal setup these data would have been correlated to actual patient responses to treatment; however, not all of the patients subjected to lung surgery will receive chemotherapy. Moreover, these chemotherapeutic interventions usually do not take place in the moment directly after surgery. Nevertheless, such data - if available - will be collected on the long-run.