Isolation and identification of adipose-derived stromal/stem cells from breast cancer patients after exposure neoadjuvant chemotherapy.

BACKGROUND: With recent research advances, adipose-derived stromal/stem cells (ASCs) have been demonstrated to facilitate the survival of fat grafts and thus are increasingly used for reconstructive procedures following surgery for breast cancer. Unfortunately, in patients, following radiation and chemotherapy for breast cancer suggest that these cancer treatment therapies may limit stem cell cellular functions important for soft tissue wound healing. For clinical translation to patients that have undergone cancer treatment, it is necessary to understand the effects of these therapies on the ASC's ability to improve fat graft survival in clinical practice. AIM: To investigate whether the impact on ASCs function capacity and recovery in cancer patients may be due to the chemotherapy. METHODS: ASCs were isolated from the cancerous side and noncancerous side of the breast from the same patients with receiving neoadjuvant chemotherapy (NAC) or not-receiving NAC. ASCs were in vitro treated with 5-fluorouracil (5-FU), doxorubicin (DXR), and cyclophosphamide (Cytoxan) at various concentrations. The stem cells yield, cell viability, and proliferation rates were measured by growth curves and MTT assays. Differentiation capacity for adipogenesis was determined by qPCR analysis of the specific gene markers and histological staining. RESULTS: No significant differences were observed between the yield of ASCs in patients receiving NAC treatment and not-receiving NAC. ASCs yield from the cancerous side of the breast showed lower than the noncancerous side of the breast in both patients receiving NAC and not-receiving NAC. The proliferation rates of ASCs from patients didn't differ much before and after NAC upon in vitro culture, and these cells appeared to retain the capacity to acquire adipocyte traits simile to the ASCs from patients not-receiving NAC. After cessation and washout of the drugs for another a week of culturing, ASCs showed a slow recovery of cell growth capacity in 5-FU-treated groups but was not observed in ASCs treated with DXR groups. CONCLUSION: Neoadjuvant therapies do not affect the functioning capacity of ASCs. ASCs may hold great potential to serve as a cell source for fat grafting and reconstruction in patients undergoing chemotherapy.

The Effect of the Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid and Paclitaxel Treatment on Full-Thickness Wound Healing in Mice.

INTRODUCTION: Neoadjuvant chemotherapy prior to lumpectomy or mastectomy for breast cancer challenges wound healing. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to work synergistically with paclitaxel in vitro and in preclinical studies. In addition, our laboratory has demonstrated that SAHA treatment decreases paclitaxel-associated stem cell toxicity, modulates inflammatory response, and promotes wound healing in injured fibroblast cells. Our goal was to determine if combined SAHA and paclitaxel treatment would improve wound healing in an in vivo full-thickness murine model, without altering antitumor effect. METHODS: Thirty-two nude athymic mice received intraperitoneal injections of paclitaxel (20 mg/kg), SAHA (25 mg/kg), paclitaxel + SAHA (20 mg/kg + 25 mg/kg), or no treatment for 2 weeks prior to surgery. Under general anesthesia, 8-mm full-thickness dorsal wounds were created in all animals, and a silicone splint was attached to minimize wound contraction. The wounds were measured twice a week with a surgical caliper until healing was complete. To evaluate the in vivo effect of drug treatment, 16 athymic nude mice with MDA-MB-231 xenografts received the treatments described previously, following which tumor volumes were compared between groups. RESULTS: Average wound healing time was prolonged in mice treated with paclitaxel (20 ± 1.9 days), and combination SAHA + paclitaxel therapy improved average wound healing time (17.0 ± 1.8 days). In the xenograft model, the antitumor effect of SAHA and paclitaxel (average tumor volume 43.9 ± 34.1 mm) was greater than paclitaxel alone (105.8 ± 73.8 mm). CONCLUSIONS: The addition of SAHA to taxane chemotherapy improves the therapeutic effect on triple-negative breast cancer while decreasing the detrimental effect of paclitaxel on wound healing. This may have substantial implications on improving outcomes in breast reconstruction following chemotherapy.

Spectral analysis of heart rate variability predicts mortality and instability from vascular injury.

BACKGROUND: Spectral analysis of continuous blood pressure and heart rate variability provides a quantitative assessment of autonomic response to hemorrhage. This may reveal markers of mortality as well as endpoints of resuscitation. METHODS: Fourteen male Yorkshire pigs, ranging in weight from 33 to 36 kg, were included in the analysis. All pigs underwent laparotomy and then sustained a standardized retrohepatic inferior vena cava injury. Animals were then allowed to progress to class 3 hemorrhagic shock and where then treated with abdominal sponge packing followed by 6 h of crystalloid resuscitation. If the pigs survived the 6 h resuscitation, they were in the survival (S) group, otherwise they were placed in the nonsurvival (NS) group. Fast Fourier transformation calculations were used to convert the components of blood pressure and heart rate variability into corresponding frequency classifications. Autonomic tones are represented as the following: high frequency (HF) = parasympathetic tone, low frequency (LF) = sympathetic, and very low frequency (VLF) = renin-angiotensin aldosterone system. The relative sympathetic to parasympathetic tone was expressed as LF/HF ratio. RESULTS: Baseline hemodynamic parameters were equal for the S (n = 11) and NS groups. LF/HF was lower at baseline for the NS group but was higher after hemorrhage and the resuscitation period indicative of a predominately parasympathetic response during hemorrhagic shock before mortality. HF signal was lower in the NS group during the resuscitation indicating a relatively lower sympathetic tone during hemorrhagic shock, which may have contributed to mortality. Finally, the NS group had a lower VLF signal at baseline (e.g., [S] 16.3 ± 2.5 versus [NS] 4.6 ± 2.9 P < 0.05,) which was predictive of mortality and hemodynamic instability in response to a similar hemorrhagic injury. CONCLUSIONS: An increased LF/HF ratio, indicative of parasympathetic predominance following injury and during resuscitation of hemorrhagic shock was a marker of impending death. Spectral analysis of heart rate variability can also identify autonomic lability following hemorrhagic injuries with implications for first responder triage. Furthermore, a decreased VLF signal at baseline indicates an additional marker of hemodynamic instability and marker of mortality following a hemorrhagic injury. These data indicate that continuous quantitative assessment of autonomic response can be a predictor of mortality and potentially guide resuscitation of patients in hemorrhagic shock.

The endothelial cell secretome as a novel treatment to prime adipose-derived stem cells for improved wound healing in diabetes.

BACKGROUND: Chronic wounds are a common surgical problem exacerbated by diabetes and ischemia. Although adipose-derived stem cells (ASCs) have shown promise as a wound healing therapy, their function and proliferation are hindered in diabetes. This study examines the ability of the human umbilical vein endothelial cell (HUVEC) secretome to reverse the deleterious effects of high glucose concentrations on ASCs through priming, thereby enhancing their ability to participate in angiogenesis and wound healing. METHODS: Institutional review board-approved human ASCs were cultured in M199 medium with or without glucose (30 mmol/L). HUVEC were grown in 30 mmol/L glucose-containing M199 medium; the resulting conditioned medium (HUVEC-CM) was collected every 3 days and used to prime ASCs. An aliquot of HUVEC-CM was heated (85°C for 30 minutes) to produce thermal denaturation of protein. Viability, proliferation, and endothelial differentiation were measured by MTT assays, growth curves, and quantitative polymerase chain reaction, respectively. A Matrigel assay was used to assess the ability of primed ASCs to participate in capillary-like tube formation. An Institutional Animal Care and Use Committee-approved in vivo murine model of diabetic and ischemic hindlimbs was used to evaluate the angiogenic potential of primed stem cells. Human ASCs were cultured with either control M199 or HUVEC-CM. Mice were randomized to a control group, an unprimed ASC group, or a HUVEC-primed ASC group. Cellular therapies were injected into the ischemic muscle. Thirty days later, slides were made. Microvessels were counted by three blinded observers. RESULTS: MTT assays revealed that HUVEC-priming induced a 1.5 times increase in cell viability over diabetic controls. This promoting effect was lost with heated HUVEC-CM (P < .001), indicating that the active molecules are of protein origin. After 9 days, ASCs cultured in 30 mmol/L glucose solution showed a 14% reduction in growth from nondiabetic controls (P = .013) and exhibited atrophic morphology. Conversely, diabetic HUVEC-primed stem cells demonstrated a nearly four-fold increase in proliferation (P < .05) and took on a fusiform, endothelial-like phenotype. Polymerase chain reaction demonstrated enhanced expression of CD31 messenger RNA by 4.7-fold after 14 days in the HUVEC-primed group, and endothelial nitric oxide synthase messenger RNA messenger RNA was increased 20.1-fold from controls. Unlike unprimed controls, HUVEC-primed ASCs readily formed capillary-like tube networks on Matrigel. Diabetic mice that were injected with HUVEC-primed ASCs demonstrated greater vessel density than both controls (2.1-fold) and unprimed stem cell treatments (P < .001). CONCLUSIONS: HUVECs secrete protein factors that significantly increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to enhanced angiogenesis.

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

INTRODUCTION: Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. METHODS: Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 µM) or with low-dose paclitaxel (0.1 µM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. RESULTS: Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. CONCLUSIONS: Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Kaolin-based hemostatic dressing improves hemorrhage control from a penetrating inferior vena cava injury in coagulopathic swine.

BACKGROUND: Retrohepatic inferior vena cava (RIVC) injuries are often lethal due to challenges in obtaining hemorrhage control. We hypothesized that packing with a new kaolin-based hemostatic dressing (Control+; Z-Medica, Wallingford, CT) would improve hemorrhage control from a penetrating RIVC injury compared with packing with standard laparotomy sponges alone. METHODS: Twelve male Yorkshire pigs received a 25% exchange transfusion of blood for refrigerated normal saline to induce a hypothermic coagulopathy. A laparotomy was performed and a standardized 1.5 cm injury to the RIVC was created which was followed by temporary abdominal closure and a period of uncontrolled hemorrhage. When the mean arterial pressure reached 70% of baseline, demonstrating hemorrhagic shock, the abdomen was re-entered, and the injury was treated with perihepatic packing using standard laparotomy sponges (L; n = 6) or a new kaolin-based hemostatic dressing (K; n = 6). Animals were then resuscitated for 6 hours with crystalloid solution. The two groups were compared using the Wilcoxon rank sum test and Fisher exact test. A p value of 0.05 or less was considered statistically significant. RESULTS: There was no difference in the animal's temperature, heart rate, mean arterial pressure, cardiac output, and blood loss at baseline or before packing was performed (all p > 0.05). In the laparotomy sponge group, five of six pigs survived the entire study period, whereas all six pigs treated with kaolin-based D2 hemostatic dressings survived. Importantly, there was significantly less blood loss after packing with the new hemostatic kaolin-based dressing compared with packing with laparotomy sponge (651 ± 180 mL vs. 1073 ± 342 mL; p ≤ 0.05). CONCLUSION: These results demonstrate that the use of this new hemostatic kaolin-based dressing improved hemorrhage control and significantly decreased blood loss in this penetrating RIVC model. LEVEL OF EVIDENCE: This is basic science research based on a large animal model, level V.