Glycocalyx

All procedures performed were protocols approved by the University of Texas Houston Medical School Animal Welfare Committee. The experiments were conducted in compliance with the National Institutes of Health guidelines on the use of laboratory animals. All animals were housed at constant room temperature with a 12:12-h light-dark cycle with access to food and water ad libitum. Male C57BL/6J mice, 8–10 weeks of age were used for all experiments. To mimic the clinical scenario of trauma-induced coagulopathy in patients in shock, the coagulopathic mouse model of trauma-hemorrhagic shock described by Cohen et al was employed.^{15 (link)} In brief, under isoflurane anesthesia, a 2cm midline laparotomy incision was made, organs inspected, and the incision closed. The bilateral femoral arteries were cannulated for continuous hemodynamic monitoring and blood withdrawal or resuscitation, respectively. After 10-minute period of equilibration, mice were bled to a mean arterial pressure (MAP) of 35±5 mmHg and maintained for 90 minutes. Shams underwent anesthesia and placement of catheters but were not subjected to hemorrhagic shock. Similar to Cohen et al, mouse were coagulopathic with a PT 12.1± 0.6 after hemorrhagic shock vs. 7.5 ± 0.2 sham, p=0.02. Mice were resuscitated over the next 15 minutes with either lactated Ringer’s at 3X shed blood volume^{16 (link)} or fresh frozen plasma at 1X shed blood volume and compared to animals that underwent shock alone. At the conclusion of resuscitation, vascular catheters were removed, incisions closed, and the animals were awoken from anesthesia. After three hours, animals were sacrificed by exsanguination under isoflurane anesthesia. Blood was obtained at the time of sacrifice and lungs harvested for further analysis. The three hour time point was chosen based on our previous investigation showing that the endothelial glycocalyx was being restored by three hours of resuscitation.^{9 (link)}

The process of generating PDX models in mice from fresh primary or metastatic human cancer is extensively described in the literature (10 , 13 (link)). While individual groups have developed specific methodological approaches, the fundamentals are common. Table 1 provides a summary of approaches used to generate the most comprehensive PDX collections currently available. Briefly, pieces of primary or metastatic solid tumors maintained as tissue structures are collected by surgery or biopsy procedures. Some studies have also used fluid drained from malignant ascites or pleural effusions. Tumors are implanted as pieces or single cell suspensions, either alone or in some studies coated with matrigel or mixed with human fibroblasts or mesenchymal stem cells. The most common site of implantation is on the dorsal region of mice (subcutaneous implantation), although implantation in the same organ as the original tumor may be an option (orthotopic implantation, i.e. pancreas, oral cavity, ovary, mammary fat pad, brain, etc.). In addition, independently of the tumor origin, several approaches have implanted primary tumors in the renal capsule in an effort to increase engraftment success rates. A variety of mouse strains having different degrees of immunosuppression have been used in these studies. Supplementary Table 1 lists the principal characteristics of the most commonly used mouse strains including their level of immune suppression as well as advantages or disadvantages. For hormone sensitive tumors, some studies have used hormone supplementation with the intent of increasing engraftment rates.
Some approaches may have theoretical advantages with regard to higher and faster engraftment rates and generation of models that better recapitulate human tumors and are, therefore, more predictive. However, it is important to mention that very few studies have properly addressed comparative implantation methods for these endpoints. Studies in which PDX models have been generated simultaneously from primary tumors and metastatic lesions suggest that metastases have a higher engraftment rate (14 (link), 15 ). Defining the most appropriate host mouse strains to generate PDX models is an important consideration. It is assumed that more severely immunosuppressed models such as non-obese diabetic/severe combined immunodeficiency disorder (NOD/SCID) or NOD/SCID/IL2λ-receptor null (NSG) models are better suited for PDX generation due to higher engraftment rates. Indeed, these are the preferred rodent strains for many groups. However, in human breast cancer (HBC) where this question has been robustly interrogated, implantation in NOD/SCID versus NSG mice yielded similar take rates (16 (link)). In addition, host supplementation with estradiol pellets increased engraftment rates from 2.6 to 21.4 % while, for reasons that are unclear, co-implantation with immortalized human fibroblasts decreased engraftment rate (16 (link)). In contrast, in another study, a mixture of irradiated and non-irradiated human fibroblasts provided improved results (17 (link)). Likewise orthotopic tumor implantation (“orthoxenografts”, (18 (link))) may also confer a translational advantage, as the tumor develops in the same anatomical microenvironment. Generation of orthoxenografts is more labor-intensive, requires complex surgery, is more expensive and often requires imaging methods to monitor tumor growth. However, for several tumor types (e.g. ovarian cancer or lung cancer), this approach substantially increases tumor take rates (19 (link)). In this vein, orthotopic implantation in the testis is essential for the growth of testicular germ cell tumors. As for tumor implantation in the renal capsule, it yielded an impressive 90 % engraftment rate in non-small cell lung cancer (NSCLC) as compared to 25% following subcutaneous implantation, although these results were not obtained from a single comparative study (20 (link), 21 (link)). Furthermore, renal cell capsule implantation shortens time to engraftment, which is one of the most important variables for studies seeking to implement real time PDX data for personalized cancer treatment (20 (link)).