Study tumor homing mechanisms of macrophages using intravital microscopy
Morbidity and subsequent mortality in cancer patients is often the result of metastases. To be able to more specifically target these metastases, we are trying to deliver genetic constructs (therapeutic DNA/RNA) to specific sites of cancer-associated inflammation using leukocytes. We hypothesize that CSF1R inhibition can improve the therapeutic effects of administered leukocytes through reducing the number of pro-tumor macrophages residing in the tumor. We have already macroscopically determined the extent to which systemically injected white blood cells are recruited to tumors. Using the intravital microscopy technology, we hope to acquire a further understanding of this tumor-targeted migration on a microscopic level. By obtaining and publishing this data, we will contribute important insights on cell migration that is relevant for the field of cell therapy, gene therapy and immunotherapy in general and by doing so, help advance these strategies to the clinic. For example, the recent success of CAR T cell therapy for B cell malignancy cannot easily be translated to solid tumors. This is, in part, a consequence of the lack of understanding of T cell recruitment to solid tumors.
Implanting and growing small-sized tumors (average diameter of 4-5 mm) in mice is not very distressful for the animals since this size of tumors does not impact the movement and behavior of the animals and tumor metastasis only occurs in a much later stage. Furthermore, all manipulations (e.g. injections) occur under general anesthesia. No additional distress is to be expected from imaging the tumor vasculature since these are endpoint experiments in which the animals are killed after imaging while still under anesthesia. Some are implanted with an imaging device on their back, which is normally well-tolerated, but can be stressful in some cases. Careful monitoring of animal behavior and signs of infection by trained personnel is frequently performed and in case of distress, pain relief (buprenorphine) is provided.
In total, these experiments involve the use of 80 mice. This number of animals is necessary to ensure that the obtained results correctly represent the effect of the performed procedure. Interactions of circulating immune cells with endothelial cells involves many adherence mechanisms and is further complicated by interactions with other cells and passing factors in the blood as well as shear stress caused by the blood flow. Although individual components of these interactions can be investigated in vitro, we have to resort to the use of real animals to study this complex cascade of events in total. To reduce the number of animals, we use ex vivo cell cultures to amplify the number of donor cells from a minimal amount of donor animals. For the mice that will be implanted with dorsal window chambers, special plastic light-weight chambers (instead of heavier and larger titanium chambers) were developed to minimise discomfort. In addition, proper environmental enrichment is present throughout the experiments.
Implanting and growing small-sized tumors (average diameter of 4-5 mm) in mice is not very distressful for the animals since this size of tumors does not impact the movement and behavior of the animals and tumor metastasis only occurs in a much later stage. Furthermore, all manipulations (e.g. injections) occur under general anesthesia. No additional distress is to be expected from imaging the tumor vasculature since these are endpoint experiments in which the animals are killed after imaging while still under anesthesia. Some are implanted with an imaging device on their back, which is normally well-tolerated, but can be stressful in some cases. Careful monitoring of animal behavior and signs of infection by trained personnel is frequently performed and in case of distress, pain relief (buprenorphine) is provided.
In total, these experiments involve the use of 80 mice. This number of animals is necessary to ensure that the obtained results correctly represent the effect of the performed procedure. Interactions of circulating immune cells with endothelial cells involves many adherence mechanisms and is further complicated by interactions with other cells and passing factors in the blood as well as shear stress caused by the blood flow. Although individual components of these interactions can be investigated in vitro, we have to resort to the use of real animals to study this complex cascade of events in total. To reduce the number of animals, we use ex vivo cell cultures to amplify the number of donor cells from a minimal amount of donor animals. For the mice that will be implanted with dorsal window chambers, special plastic light-weight chambers (instead of heavier and larger titanium chambers) were developed to minimise discomfort. In addition, proper environmental enrichment is present throughout the experiments.