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Complex spatial dynamics of oncolytic viruses in vitro: mathematical and experimental approaches.

Wodarz D, Hofacre A, Lau JW, Sun Z, Fan H, Komarova NL - PLoS Comput. Biol. (2012)

Bottom Line: We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run.The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success.Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, University of California, Irvine, California, United States of America. dwodarz@uci.edu

ABSTRACT
Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call "hollow ring structure", "filled ring structure", and "disperse pattern". An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built.

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Related in: MedlinePlus

Observed patterns of AdEGFPuci infection in HEK293-mCherry cells.(A) HEK293-mCherry cells were infected at an MOI≪1 and tracked every 24 h beginning at 5 days post-infection when initial spread of infection had occurred. Three representative patterns of AdEGFPuci infection were observed after at least 13 days post infection (pattern (i)) or as long as 19 dpi (patterns (ii) and (iii)) as shown in the micrographs (100×). The left panels represent the detection of HEK293-mCherry cell nuclei in the culture (mCherry RFP), whereas the right panels depict the identical field of view of HEK293-mCherry cells viewed for green fluorescence to detect cells infected with AdEGFPuci (EGFP). (B) Limited pattern (iii) of HEK293-mCherry cells infected by AdEGFPuci. The top panels depict AdEGFPuci infected cells (EGFP-positive), the middle panels depict all the HEK293-mCherry cell nuclei in the culture (mCherry RFP), and the bottom panels is the merge of those panels, illustrating infected vs. uninfected cells. The panels on the left are micrographs taken at 100× magnification; the right panels encompass the boxed area of the left images at 200× magnification. The arrows in the right panels point to an AdEGFPuci infected cell (inf.) and an uninfected AdEGFPuci cell (un inf.) within the center of the virus infected region of HEK293-mCherry cells at 19 dpi. The scale bar in both Figure 1A and 1B for the 100× magnifications represents 671.12 uM, the 200× image scale bar is 335.48 uM. The scale bar in the 100× magnification defines the region of each micrograph needed to capture the area of the infected cells of an individual infection (compare Figure 1A pattern (i) with patterns (ii) and (iii)).
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pcbi-1002547-g001: Observed patterns of AdEGFPuci infection in HEK293-mCherry cells.(A) HEK293-mCherry cells were infected at an MOI≪1 and tracked every 24 h beginning at 5 days post-infection when initial spread of infection had occurred. Three representative patterns of AdEGFPuci infection were observed after at least 13 days post infection (pattern (i)) or as long as 19 dpi (patterns (ii) and (iii)) as shown in the micrographs (100×). The left panels represent the detection of HEK293-mCherry cell nuclei in the culture (mCherry RFP), whereas the right panels depict the identical field of view of HEK293-mCherry cells viewed for green fluorescence to detect cells infected with AdEGFPuci (EGFP). (B) Limited pattern (iii) of HEK293-mCherry cells infected by AdEGFPuci. The top panels depict AdEGFPuci infected cells (EGFP-positive), the middle panels depict all the HEK293-mCherry cell nuclei in the culture (mCherry RFP), and the bottom panels is the merge of those panels, illustrating infected vs. uninfected cells. The panels on the left are micrographs taken at 100× magnification; the right panels encompass the boxed area of the left images at 200× magnification. The arrows in the right panels point to an AdEGFPuci infected cell (inf.) and an uninfected AdEGFPuci cell (un inf.) within the center of the virus infected region of HEK293-mCherry cells at 19 dpi. The scale bar in both Figure 1A and 1B for the 100× magnifications represents 671.12 uM, the 200× image scale bar is 335.48 uM. The scale bar in the 100× magnification defines the region of each micrograph needed to capture the area of the infected cells of an individual infection (compare Figure 1A pattern (i) with patterns (ii) and (iii)).

Mentions: In order to examine spatial virus spread in a relatively simple setting, we constructed a recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial (293) cells [39]. The experiment was set up such that cells are arranged in a two-dimensional layer, and virus spread is most likely to occur to neighboring cells. An agar overlay prevents long-range spread of the virus away from infected cells in the culture medium. This set-up allows us to not only quantify the number of infected cells over time, but also the spatial patterns of infected cells that are formed as the virus population expands. In addition, we used fluorescent markers to visualize the spatial distribution of all cells (infected and uninfected) by generating HEK293-H2BmCherry cells, that stably express the core nuclear histone protein H2B fused to mCherry (a highly photostable, monomeric red fluorescent protein (RFP)) [40]. Thus, using HEK293-H2BmCherry cells allows us to visualize all the cell nuclei (i.e., intact cells) in any particular culture. The culture was infected at a very low multiplicity of infection (MOI), such that any area of infection resulted from a single “founder” infected cell. Each culture contained several such founder cells that were sufficiently separated from each other, allowing us to track multiple growth foci across the dish. Details of the experimental procedures are given in the Methods section. The earliest stages of virus growth starting from a single founder infected cell were characterized in detail in a separate study [39]. This gave rise to the interesting observation that while virus extinction was a likely event as long as the number of infected cells in a given area was less than three, spreading virus growth was always observed once the number of infected cells reached three or higher. In the current study, we followed the growth of such spreading infections and characterized the consequent growth patterns. We observe three basic patterns of virus spread, which interestingly occur under identical experimental conditions and even within the same culture. They are shown in Figure 1A and described as follows. (i) In the first pattern, the virus infection spreads rapidly outwards as a ring, leaving no cells behind in the core of the ring (Figure 1A, pattern (i)). This classic plaque pattern is observed in virus growth experiments. We call this the “hollow ring” structure. In the second and third patters there is viral spread, but it is limited. (ii) In the second case, a “disperse” growth pattern is observed, where the virus population expands as a mixed cluster of infected and uninfected cells (Figure 1A, pattern (ii)). Finally, the virus population expands as a thinner ring, but in contrast to the first case, uninfected cells are left behind in the core of the ring (Figure 1A, pattern (iii)). We call this the “filled ring” structure. A limited growth pattern is magnified in Figure 1B, in which uninfected cells are visible within the center of the virus infected population. In the top right panel of Figure 1B, an AdEGFPuci infected (fluorescent) cell is indicated (arrow, inf.), whereas an uninfected cell in the center of the spreading infection does not fluoresce green (arrow, un inf.). The same cells are indicated in the middle right panel of Figure 1B, showing red fluorescence. In the bottom left panel of Figure 1B, images of the top and middle panels are merged; infected cell (arrow, inf.) fluoresces yellow, while the uninfected cell, (arrow, un inf.) remains red. As mentioned the area over which the infection spread remained limited in patterns (ii) and (iii) and persisted throughout the infection (through 19 dpi). In contrast, in pattern (i) the ring of infected cells continued to spread outward as long as there was space; cell clearing in the center of the plaque was apparent at 13 dpi, as shown in Figure 1A. Similar patterns of spreading infection were also seen in Ad293 cells, a HEK293 cell derivative optimized for adenovirus plaque assays. Overall, among 436 scored growth foci, the hollow ring structure was found in 45%, and the limited patterns in 55% of cases.


Complex spatial dynamics of oncolytic viruses in vitro: mathematical and experimental approaches.

Wodarz D, Hofacre A, Lau JW, Sun Z, Fan H, Komarova NL - PLoS Comput. Biol. (2012)

Observed patterns of AdEGFPuci infection in HEK293-mCherry cells.(A) HEK293-mCherry cells were infected at an MOI≪1 and tracked every 24 h beginning at 5 days post-infection when initial spread of infection had occurred. Three representative patterns of AdEGFPuci infection were observed after at least 13 days post infection (pattern (i)) or as long as 19 dpi (patterns (ii) and (iii)) as shown in the micrographs (100×). The left panels represent the detection of HEK293-mCherry cell nuclei in the culture (mCherry RFP), whereas the right panels depict the identical field of view of HEK293-mCherry cells viewed for green fluorescence to detect cells infected with AdEGFPuci (EGFP). (B) Limited pattern (iii) of HEK293-mCherry cells infected by AdEGFPuci. The top panels depict AdEGFPuci infected cells (EGFP-positive), the middle panels depict all the HEK293-mCherry cell nuclei in the culture (mCherry RFP), and the bottom panels is the merge of those panels, illustrating infected vs. uninfected cells. The panels on the left are micrographs taken at 100× magnification; the right panels encompass the boxed area of the left images at 200× magnification. The arrows in the right panels point to an AdEGFPuci infected cell (inf.) and an uninfected AdEGFPuci cell (un inf.) within the center of the virus infected region of HEK293-mCherry cells at 19 dpi. The scale bar in both Figure 1A and 1B for the 100× magnifications represents 671.12 uM, the 200× image scale bar is 335.48 uM. The scale bar in the 100× magnification defines the region of each micrograph needed to capture the area of the infected cells of an individual infection (compare Figure 1A pattern (i) with patterns (ii) and (iii)).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375216&req=5

pcbi-1002547-g001: Observed patterns of AdEGFPuci infection in HEK293-mCherry cells.(A) HEK293-mCherry cells were infected at an MOI≪1 and tracked every 24 h beginning at 5 days post-infection when initial spread of infection had occurred. Three representative patterns of AdEGFPuci infection were observed after at least 13 days post infection (pattern (i)) or as long as 19 dpi (patterns (ii) and (iii)) as shown in the micrographs (100×). The left panels represent the detection of HEK293-mCherry cell nuclei in the culture (mCherry RFP), whereas the right panels depict the identical field of view of HEK293-mCherry cells viewed for green fluorescence to detect cells infected with AdEGFPuci (EGFP). (B) Limited pattern (iii) of HEK293-mCherry cells infected by AdEGFPuci. The top panels depict AdEGFPuci infected cells (EGFP-positive), the middle panels depict all the HEK293-mCherry cell nuclei in the culture (mCherry RFP), and the bottom panels is the merge of those panels, illustrating infected vs. uninfected cells. The panels on the left are micrographs taken at 100× magnification; the right panels encompass the boxed area of the left images at 200× magnification. The arrows in the right panels point to an AdEGFPuci infected cell (inf.) and an uninfected AdEGFPuci cell (un inf.) within the center of the virus infected region of HEK293-mCherry cells at 19 dpi. The scale bar in both Figure 1A and 1B for the 100× magnifications represents 671.12 uM, the 200× image scale bar is 335.48 uM. The scale bar in the 100× magnification defines the region of each micrograph needed to capture the area of the infected cells of an individual infection (compare Figure 1A pattern (i) with patterns (ii) and (iii)).
Mentions: In order to examine spatial virus spread in a relatively simple setting, we constructed a recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial (293) cells [39]. The experiment was set up such that cells are arranged in a two-dimensional layer, and virus spread is most likely to occur to neighboring cells. An agar overlay prevents long-range spread of the virus away from infected cells in the culture medium. This set-up allows us to not only quantify the number of infected cells over time, but also the spatial patterns of infected cells that are formed as the virus population expands. In addition, we used fluorescent markers to visualize the spatial distribution of all cells (infected and uninfected) by generating HEK293-H2BmCherry cells, that stably express the core nuclear histone protein H2B fused to mCherry (a highly photostable, monomeric red fluorescent protein (RFP)) [40]. Thus, using HEK293-H2BmCherry cells allows us to visualize all the cell nuclei (i.e., intact cells) in any particular culture. The culture was infected at a very low multiplicity of infection (MOI), such that any area of infection resulted from a single “founder” infected cell. Each culture contained several such founder cells that were sufficiently separated from each other, allowing us to track multiple growth foci across the dish. Details of the experimental procedures are given in the Methods section. The earliest stages of virus growth starting from a single founder infected cell were characterized in detail in a separate study [39]. This gave rise to the interesting observation that while virus extinction was a likely event as long as the number of infected cells in a given area was less than three, spreading virus growth was always observed once the number of infected cells reached three or higher. In the current study, we followed the growth of such spreading infections and characterized the consequent growth patterns. We observe three basic patterns of virus spread, which interestingly occur under identical experimental conditions and even within the same culture. They are shown in Figure 1A and described as follows. (i) In the first pattern, the virus infection spreads rapidly outwards as a ring, leaving no cells behind in the core of the ring (Figure 1A, pattern (i)). This classic plaque pattern is observed in virus growth experiments. We call this the “hollow ring” structure. In the second and third patters there is viral spread, but it is limited. (ii) In the second case, a “disperse” growth pattern is observed, where the virus population expands as a mixed cluster of infected and uninfected cells (Figure 1A, pattern (ii)). Finally, the virus population expands as a thinner ring, but in contrast to the first case, uninfected cells are left behind in the core of the ring (Figure 1A, pattern (iii)). We call this the “filled ring” structure. A limited growth pattern is magnified in Figure 1B, in which uninfected cells are visible within the center of the virus infected population. In the top right panel of Figure 1B, an AdEGFPuci infected (fluorescent) cell is indicated (arrow, inf.), whereas an uninfected cell in the center of the spreading infection does not fluoresce green (arrow, un inf.). The same cells are indicated in the middle right panel of Figure 1B, showing red fluorescence. In the bottom left panel of Figure 1B, images of the top and middle panels are merged; infected cell (arrow, inf.) fluoresces yellow, while the uninfected cell, (arrow, un inf.) remains red. As mentioned the area over which the infection spread remained limited in patterns (ii) and (iii) and persisted throughout the infection (through 19 dpi). In contrast, in pattern (i) the ring of infected cells continued to spread outward as long as there was space; cell clearing in the center of the plaque was apparent at 13 dpi, as shown in Figure 1A. Similar patterns of spreading infection were also seen in Ad293 cells, a HEK293 cell derivative optimized for adenovirus plaque assays. Overall, among 436 scored growth foci, the hollow ring structure was found in 45%, and the limited patterns in 55% of cases.

Bottom Line: We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run.The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success.Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, University of California, Irvine, California, United States of America. dwodarz@uci.edu

ABSTRACT
Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call "hollow ring structure", "filled ring structure", and "disperse pattern". An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built.

Show MeSH
Related in: MedlinePlus