The invasion of malignant cells into tissue is a critical step in the progression of cancer. Whil... more The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We ident...
Advanced materials (Deerfield Beach, Fla.), Jan 16, 2015
Pore-gradient microgel arrays enable thousands of parallel high resolution single-cell protein el... more Pore-gradient microgel arrays enable thousands of parallel high resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual cross-linked hydrogels facilitate gel pore expansion after electrophoresis for efficient and uniform immunoprobing. The photo-patterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.
The diagnosis of malignant pleural effusions is an important issue in the management of malignanc... more The diagnosis of malignant pleural effusions is an important issue in the management of malignancy patients. Generally, cytologic examination is a routine diagnostic technique. However, morphological interpretation of cytology is sometimes inconclusive. Here an ancillary method named BMVC test is developed for rapid detection of malignant pleural effusion to improve the diagnostic accuracy at low cost. A simple assay kit is designed to collect living cells from clinical pleural effusion and a fluorescence probe, 3,6‐Bis(1‐methyl‐4‐vinylpyridinium) carbazole diiodide (BMVC), is used to illuminate malignant cells. The fluorescence intensity is quantitatively analyzed by ImageJ program. This method yields digital numbers for the test results without any grey zone or ambiguities in the current cytology tests due to intra‐observer and inter‐observer variability. Comparing with results from double‐blind cytologic examination, this simple test gives a good discrimination between malignant ...
Photodynamic therapy (PDT) is a promising modality for the treatment of localized tumors. To caus... more Photodynamic therapy (PDT) is a promising modality for the treatment of localized tumors. To cause cell death during PDT, singlet oxygen can be generated by energy transfer from the triplet state of a photosensitizer to molecular oxygen. Among various photosensitizers, porphyrin derivatives have been widely studied. 6] However, porphyrin-based sensitizers have drawbacks to their application in PDT that include poor chemical selectivity toward intended tissue targets and a lack of specific light wavelengths that are optimal for tissue penetration and chromophore excitation. To overcome these shortcomings of the traditional porphyrin chromophore, a number of binary compounds have been recently designed and prepared with a component linked to the porphyrin photosensitizer. The resulting conjugates are highly selective for cancer cells and absorb at longer wavelengths more suitable for tissue penetration. For example, Drain and co-workers synthesized porphyrin–saccharide conjugates to increase the uptake of the photosensitizer by specific cancer cells. Dichtel et al. synthesized a binary compound to enhance singlet oxygen generation via fluorescence resonance energy transfer (FRET) by two-photon excitation of the donor chromophore to the central porphyrin acceptor. Selectivity is a key feature in the design of photosensitizers for killing cancer cells without damaging normal cells. We recently synthesized a novel 3,6-bis-(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) compound with selectivity toward cancer cells over normal cells. 13] Specifically, the fluorescence of BMVC detected in cancer cells was found to be much stronger than that in normal cells. Moreover, BMVC has a large cross-section for two-photon absorption around 820 nm, a wavelength that is close to optimal for tissue penetration of the radiation needed to excite the photosensitizer. In addition, the windows of transparency for porphyrin derivatives in the range of 450–500 nm allow us to selectively excite BMVC. For the study reported herein, we combined these features into a binary porphyrin compound for selectivity in PDT. In this compound, two BMVC molecules are covalently linked to a central 5,10-bis-(4-hydroxyphenyl)-15,20-bis-(4-methoxyphenyl)porphyrin (PAP) molecule at the ortho positions, and we termed the resulting binary compound o-2B-P. The ortho position was chosen for covalent attachment, as it is known that compounds containing ortho-5,10-disubstituted water-soluble trimethylaminophenyl groups exhibit better PDT efficacy than those with para-5,15-disubstitution. The structure of o-2B-P is shown in Figure 1 A, and details of its synthesis can be found elsewhere (Supporting Information 1). We examined the efficiency of energy transfer from the excited state of BMVC to the porphyrin in o-2B-P, as well as the efficacy of singlet oxygen generation by the porphyrin in the binary compound. The absorption and fluorescence spectra of o-2B-P, PAP, BMVC, and a mixture of BMVC and PAP in DMSO are depicted in Figures 1 B and C, respectively. The absorption of o-2B-P is almost identical to that of a mixture of BMVC and PAP. The absorption is a linear sum of the absorbances of BMVC and PAP individually, indicating that there is no appreciable interaction between the two chromophores in the ground electronic states. In contrast, the fluorescence of BMVC in o-2BP is almost totally quenched, whereas the fluorescence of PAP is enhanced by at least fivefold upon excitation of o-2B-P at 470 nm. Evidently, there is efficient energy transfer from the excited state of BMVC to PAP. To evaluate the effect of photoinduced formation of singlet oxygen by o-2B-P, we applied a photochemical method using a singlet oxygen quencher, 1,3-diphenylisobenzofuran (DPBF), to verify the generation of singlet oxygen. Because the cationic meso-tetra-(4-N-methylpyridyl)porphyrin (TMPyP) molecule is itself a singlet oxygen photosensitizer, 20] we compared the yield of singlet oxygen upon irradiation of o-2B-P and TMPyP at various wavelengths. Figure 1 D shows a comparison of the absorbance of DPBF at 417 nm in the presence of o-2B-P and TMPyP in DMSO as a function of irradiation time at 470 10 nm as well as lex>580 nm selectively from a halogen lamp. Although singlet oxygen generation by o-2B-P is slightly lower than that of TMPyP at lex>580 nm, it is the singlet oxygen generated by o-2B-P, not by TMPyP, upon excitation at 470 10 nm that provides a more useful measure of the selectivity for PDT efficacy. Despite the large number of porphyrin derivatives in blood and tissues, the porphyrin absorbance at 470 10 nm is almost negligible. Accordingly, the 450–500 nm transparent windows of porphyrin derivatives [a] C.-C. Kang, Dr. C.-C. Cho, Y.-C. Lin, Prof. C.-C. Chang, Prof. T.-C. Chang Institute of Atomic and Molecular Sciences, Academia Sinica P.O. Box 23-166, Taipei 106 (Taiwan, ROC) Fax: (+ ) 886-2-23620200 E-mail : [email protected] [b] C.-C. Kang Taiwan…
Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BMVC, 3,6-bis(1-met... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BMVC, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide, is a novel organic molecule with special tumor recognition characteristic. BMVC appears bright fluorescence in the nucleus of cancer cells while only weak fluorescence in the cytoplasm of normal cells. The strong fluorescence of BMVC in the nucleus is mainly due to significant enhancement of BMVC fluorescence upon binding to DNA. Therefore, these distinct properties of BMVC in cells allow us to differentiate cancer cells from normal cells. It appears that BMVC is a potential fluorescent tumor marker. It is of interest to elucidate the reason why BMVC can act as a potential fluorescence tumor marker. Confocal microscopic images by merging with organelle trackers suggested that BMVC mainly localizes in the nucleus and mitochondria of cancer cells, but in the lysosome of normal cells. Several leading experiments suggested that the major pathway for BMVC uptake is endocytosis. In addition, the carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and microinjection experiments showed that the destination of endocytosed BMVC is trapped in the lysosome of normal cells. However, it is not clear why BMVC is not trapped in the lysosome of cancer cells. Since the lysosomal membrane permeabilization (LMP) could sensitize cancer cell to death, we performed the cathepsin immunofluorescence and vacuolization experiments to elucidate whether there is LMP difference between cancer and normal cells. Our results showed that the LMP indeed plays an important role for trapping BMVC in the lysosome of normal cells, but releasing BMVC to the nucleus and mitochondria of cancer cells. Furthermore, the structure localization relationship (SLR) studies show that BMVC derivatives with large hydrogen bonding capacity (HBC) are less lysosomal membrane permeable in normal cells. In addition, we found the more lipophilic of BMVC derivatives the more mitochondria localization in cancer cells. It is likely that the HBC and the lipophilicity are two important characters in determining intracellular localization of BMVC derivatives in cells. In summary, the LMP and SLR studies of BMVC and its derivatives demonstrated a new direction for further design and synthesis of better fluorescent tumor markers and also provided a new platform for cancer selective drug through the combination of a cancer targeting molecule and anticancer drug. Reference: [1] C. C. Kang, C. C. Chang, T. C. Chang, L. J. Liao, P. J. Lou, W. Xie, E. S. Yeung, Analyst 2007, 132, 745. [2] L. J. Liao, C. C. Kang, I. S. Jan, H. C. Chen, C. L. Wang, P. J. Lou, T. C. Chang, Analyst 2009, 134, 708. [3] N. Fehrenbacher, M. Jaattela, Cancer Res 2005, 65, 2993. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4626.
The invasion of malignant cells into tissue is a critical step in the progression of cancer. Whil... more The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We ident...
Advanced materials (Deerfield Beach, Fla.), Jan 16, 2015
Pore-gradient microgel arrays enable thousands of parallel high resolution single-cell protein el... more Pore-gradient microgel arrays enable thousands of parallel high resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual cross-linked hydrogels facilitate gel pore expansion after electrophoresis for efficient and uniform immunoprobing. The photo-patterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.
The diagnosis of malignant pleural effusions is an important issue in the management of malignanc... more The diagnosis of malignant pleural effusions is an important issue in the management of malignancy patients. Generally, cytologic examination is a routine diagnostic technique. However, morphological interpretation of cytology is sometimes inconclusive. Here an ancillary method named BMVC test is developed for rapid detection of malignant pleural effusion to improve the diagnostic accuracy at low cost. A simple assay kit is designed to collect living cells from clinical pleural effusion and a fluorescence probe, 3,6‐Bis(1‐methyl‐4‐vinylpyridinium) carbazole diiodide (BMVC), is used to illuminate malignant cells. The fluorescence intensity is quantitatively analyzed by ImageJ program. This method yields digital numbers for the test results without any grey zone or ambiguities in the current cytology tests due to intra‐observer and inter‐observer variability. Comparing with results from double‐blind cytologic examination, this simple test gives a good discrimination between malignant ...
Photodynamic therapy (PDT) is a promising modality for the treatment of localized tumors. To caus... more Photodynamic therapy (PDT) is a promising modality for the treatment of localized tumors. To cause cell death during PDT, singlet oxygen can be generated by energy transfer from the triplet state of a photosensitizer to molecular oxygen. Among various photosensitizers, porphyrin derivatives have been widely studied. 6] However, porphyrin-based sensitizers have drawbacks to their application in PDT that include poor chemical selectivity toward intended tissue targets and a lack of specific light wavelengths that are optimal for tissue penetration and chromophore excitation. To overcome these shortcomings of the traditional porphyrin chromophore, a number of binary compounds have been recently designed and prepared with a component linked to the porphyrin photosensitizer. The resulting conjugates are highly selective for cancer cells and absorb at longer wavelengths more suitable for tissue penetration. For example, Drain and co-workers synthesized porphyrin–saccharide conjugates to increase the uptake of the photosensitizer by specific cancer cells. Dichtel et al. synthesized a binary compound to enhance singlet oxygen generation via fluorescence resonance energy transfer (FRET) by two-photon excitation of the donor chromophore to the central porphyrin acceptor. Selectivity is a key feature in the design of photosensitizers for killing cancer cells without damaging normal cells. We recently synthesized a novel 3,6-bis-(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) compound with selectivity toward cancer cells over normal cells. 13] Specifically, the fluorescence of BMVC detected in cancer cells was found to be much stronger than that in normal cells. Moreover, BMVC has a large cross-section for two-photon absorption around 820 nm, a wavelength that is close to optimal for tissue penetration of the radiation needed to excite the photosensitizer. In addition, the windows of transparency for porphyrin derivatives in the range of 450–500 nm allow us to selectively excite BMVC. For the study reported herein, we combined these features into a binary porphyrin compound for selectivity in PDT. In this compound, two BMVC molecules are covalently linked to a central 5,10-bis-(4-hydroxyphenyl)-15,20-bis-(4-methoxyphenyl)porphyrin (PAP) molecule at the ortho positions, and we termed the resulting binary compound o-2B-P. The ortho position was chosen for covalent attachment, as it is known that compounds containing ortho-5,10-disubstituted water-soluble trimethylaminophenyl groups exhibit better PDT efficacy than those with para-5,15-disubstitution. The structure of o-2B-P is shown in Figure 1 A, and details of its synthesis can be found elsewhere (Supporting Information 1). We examined the efficiency of energy transfer from the excited state of BMVC to the porphyrin in o-2B-P, as well as the efficacy of singlet oxygen generation by the porphyrin in the binary compound. The absorption and fluorescence spectra of o-2B-P, PAP, BMVC, and a mixture of BMVC and PAP in DMSO are depicted in Figures 1 B and C, respectively. The absorption of o-2B-P is almost identical to that of a mixture of BMVC and PAP. The absorption is a linear sum of the absorbances of BMVC and PAP individually, indicating that there is no appreciable interaction between the two chromophores in the ground electronic states. In contrast, the fluorescence of BMVC in o-2BP is almost totally quenched, whereas the fluorescence of PAP is enhanced by at least fivefold upon excitation of o-2B-P at 470 nm. Evidently, there is efficient energy transfer from the excited state of BMVC to PAP. To evaluate the effect of photoinduced formation of singlet oxygen by o-2B-P, we applied a photochemical method using a singlet oxygen quencher, 1,3-diphenylisobenzofuran (DPBF), to verify the generation of singlet oxygen. Because the cationic meso-tetra-(4-N-methylpyridyl)porphyrin (TMPyP) molecule is itself a singlet oxygen photosensitizer, 20] we compared the yield of singlet oxygen upon irradiation of o-2B-P and TMPyP at various wavelengths. Figure 1 D shows a comparison of the absorbance of DPBF at 417 nm in the presence of o-2B-P and TMPyP in DMSO as a function of irradiation time at 470 10 nm as well as lex>580 nm selectively from a halogen lamp. Although singlet oxygen generation by o-2B-P is slightly lower than that of TMPyP at lex>580 nm, it is the singlet oxygen generated by o-2B-P, not by TMPyP, upon excitation at 470 10 nm that provides a more useful measure of the selectivity for PDT efficacy. Despite the large number of porphyrin derivatives in blood and tissues, the porphyrin absorbance at 470 10 nm is almost negligible. Accordingly, the 450–500 nm transparent windows of porphyrin derivatives [a] C.-C. Kang, Dr. C.-C. Cho, Y.-C. Lin, Prof. C.-C. Chang, Prof. T.-C. Chang Institute of Atomic and Molecular Sciences, Academia Sinica P.O. Box 23-166, Taipei 106 (Taiwan, ROC) Fax: (+ ) 886-2-23620200 E-mail : [email protected] [b] C.-C. Kang Taiwan…
Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BMVC, 3,6-bis(1-met... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BMVC, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide, is a novel organic molecule with special tumor recognition characteristic. BMVC appears bright fluorescence in the nucleus of cancer cells while only weak fluorescence in the cytoplasm of normal cells. The strong fluorescence of BMVC in the nucleus is mainly due to significant enhancement of BMVC fluorescence upon binding to DNA. Therefore, these distinct properties of BMVC in cells allow us to differentiate cancer cells from normal cells. It appears that BMVC is a potential fluorescent tumor marker. It is of interest to elucidate the reason why BMVC can act as a potential fluorescence tumor marker. Confocal microscopic images by merging with organelle trackers suggested that BMVC mainly localizes in the nucleus and mitochondria of cancer cells, but in the lysosome of normal cells. Several leading experiments suggested that the major pathway for BMVC uptake is endocytosis. In addition, the carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and microinjection experiments showed that the destination of endocytosed BMVC is trapped in the lysosome of normal cells. However, it is not clear why BMVC is not trapped in the lysosome of cancer cells. Since the lysosomal membrane permeabilization (LMP) could sensitize cancer cell to death, we performed the cathepsin immunofluorescence and vacuolization experiments to elucidate whether there is LMP difference between cancer and normal cells. Our results showed that the LMP indeed plays an important role for trapping BMVC in the lysosome of normal cells, but releasing BMVC to the nucleus and mitochondria of cancer cells. Furthermore, the structure localization relationship (SLR) studies show that BMVC derivatives with large hydrogen bonding capacity (HBC) are less lysosomal membrane permeable in normal cells. In addition, we found the more lipophilic of BMVC derivatives the more mitochondria localization in cancer cells. It is likely that the HBC and the lipophilicity are two important characters in determining intracellular localization of BMVC derivatives in cells. In summary, the LMP and SLR studies of BMVC and its derivatives demonstrated a new direction for further design and synthesis of better fluorescent tumor markers and also provided a new platform for cancer selective drug through the combination of a cancer targeting molecule and anticancer drug. Reference: [1] C. C. Kang, C. C. Chang, T. C. Chang, L. J. Liao, P. J. Lou, W. Xie, E. S. Yeung, Analyst 2007, 132, 745. [2] L. J. Liao, C. C. Kang, I. S. Jan, H. C. Chen, C. L. Wang, P. J. Lou, T. C. Chang, Analyst 2009, 134, 708. [3] N. Fehrenbacher, M. Jaattela, Cancer Res 2005, 65, 2993. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4626.
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