Raman microspectroscopy allows probing subcellular compartments and provides a unique spectral fingerprint indicative of endogenous molecular composition. Although several spectroscopic cell studies have been reported on fixed samples, only few attempts concern single growing cells. Here, we have tested different optical substrates that would best preserve cell integrity and allow direct measurement of Raman spectra at the single living cell level. Calu-1 lung cancer cells were used as a model and their morphology and growth were assessed on Raman substrates like quartz, calcium fluoride, and zinc selenide. Data show that quartz was the most appropriate taking into consideration both cell morphology and proliferation rate (47% on quartz vs. 55% of BrdU-positive cells on conventional plastic). Using quartz, 40 cells were analysed and Raman spectra were collected from nuclei and cytoplasms using a 785 nm laser excitation of 30 mW at the sample, in the spectral range of 580-1750 cm(-1), and an acquisition time of 2 x 10 sec/spectrum. Discriminant spectral information related to nucleus and cytoplasm were extracted by multivariate statistical methods and attributed to nucleic acids, lipids, and proteins. Finally, Raman spectral imaging was performed to show the distribution of these components within the cell.

BackgroundThe cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment.MethodsTo this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation.ResultsWe show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected β1 integrin expression nor the state of phosphorylation of FAK and RhoA.ConclusionThis study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

Tumour drug-resistant ABCB1 gene expression is regulated at the chromatin level through epigenetic mechanisms. We examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on ABCB1 gene expression in small cell lung carcinoma (SCLC) drugsensitive (H69WT) or etoposide-resistant (H69VP) cells. We found that TSA induced an increase in ABCB1 expression in drugsensitive cells, but strongly decreased it in drug-resistant cells. These up-and downregulations occurred at the transcriptional level. Protein synthesis inhibition reduced these modulations, but did not completely suppress them. Differential temporal patterns of histone acetylation were observed at the ABCB1 promoter: increase in H4 acetylation in both cell lines, but different H3 acetylation with a progressive increase in H69WT cells but a transient one in H69VP cells. ABCB1 regulations were not related with the methylation status of the promoter À50GC, À110GC, and Inr sites, and did not result in further changes to these methylation profiles. Trichostatin A treatment did not modify MBD1 binding to the ABCB1 promoter and similarly increased PCAF binding in both H69 cell lines. Our results suggest that in H69 drug-resistant SCLC cell line TSA induces downregulation of ABCB1 expression through a transcriptional mechanism, independently of promoter methylation, and MBD1 or PCAF recruitment.

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. A n increasing body of evidence indicates that the tumor cell microenvironment is a key determinant of cell response to cytotoxic drugs. The environmental causes that contribute to cancer cell survival after initial therapy, allowing resistant cells to proliferate are multifactorial. They include particularly limited drug penetration, tumor cell adaptation to hypoxia, acidic extracellular pH, and direct contact between cancer cells and the ECM or adjacent cells.(1) Concerning ECM proteins, fibronectin or collagen type I and IV have been shown to have the potential to confer resistance to diverse chemotherapeutic agents in cancer cell lines originated from both hematopoietic and solid tumors. (2,3) This has led to the concept of CAM-DR, a form of de novo drug resistance which may play an important role in the acquisition of a multidrug resistance phenotype. (4,5) Studies on the impact of the microenvironment on drug response have focused exclusively on the cytotoxic or proapoptotic effects induced by an extensive panel of well-known chemotherapeutics, either microtubule-disturbing agents such as vinca-alcaloids, taxoids, or DNA-damaging compounds such as nitrogen mustards and anthracyclines. (6,7) However, with chemotherapeutics such as anthracyclines, beyond their well-known antitumor activity, it has been recently demonstrated that they may display potent anti-invasive effects. Indeed, doxorubicin has been shown to inhibit cell escape from multicellular spheroids with cells of prostate, colon, breast, or lung carcinoma.(8) Using the highly mobile fibrosarcoma cell line HT1080 it has been shown that DA-125, a new analog of doxorubicin, inhibits the invasion of these cells by downregulating MMP.(9...