PGC-1α drives small cell neuroendocrine cancer progression towards an ASCL1-expressing subtype with increased mitochondrial capacity.

Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers comprised of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of PGC-1α, a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNC types. PGC-1α correlated tightly with increased expression of the lineage marker ASCL1 through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. PGC-1α overexpression enhanced OXPHOS, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These findings reveal the metabolic heterogeneity among SCNC subtypes and identify PGC-1α-induced OXPHOS as a regulator of SCNC lineage plasticity.

Inability of ovarian cancers to upregulate their MHC-class I surface expression marks their aggressiveness and increased susceptibility to NK cell-mediated cytotoxicity.

We extended our previous observations with other tumor models to study seven ovarian tumor cell lines-OVCAR3, OVCAR4, OVCAR8, SKOV3, Kuramochi, OAW28, and CaOV3. We found that NK cells targeted and killed poorly differentiated OVCAR8 and CAOV3; these two tumor lines express lower MHC-class I and higher CD44 surface receptors. OVCAR3 and OVCAR4 were more resistant to NK cell-mediated cytotoxicity, and SKOV3, Kuramochi and OAW28 had intermediate sensitivity to NK cell-mediated cytotoxicity, likely representing well-differentiated and moderately differentiated ovarian tumor cell lines, respectively. Similar trends were observed for secretion of IFN-γ by the NK cells when co-cultured with different ovarian tumor cell lines. Treatment with both IFN-γ and TNF-α upregulated MHC-class I in all ovarian tumor cell lines and resulted in tumor resistance to NK cell-mediated cytotoxicity and decreased secretion of IFN-γ in co-cultures of NK cells with tumors cells with the exception of OVCAR8 and CAOV3 which did not upregulate MHC-class I and remained sensitive to NK cell-mediated cytotoxicity and increased secretion of IFN-γ when co-cultured with NK cells. Similarly, treatment with NK cell supernatants induced resistance to NK cell-mediated cytotoxicity in OVCAR4 but not in OVCAR8, and the resistance to killing was correlated with the increased surface expression of MHC-class I in OVCAR4 but not in OVCAR8. In addition, OVCAR4 was found to be carboplatin sensitive before and after treatment with IFN-γ and NK cell supernatants, whereas OVCAR8 remained carboplatin resistant with and without treatment with IFN-γ and NK cell supernatants. Overall, sensitivity to NK cell-mediated killing correlated with the levels of tumor differentiation and aggressiveness, and more importantly, poorly differentiated ovarian tumors were unable to upregulate MHC-class I under the activating conditions for MHC-class I, a feature that was not seen in other tumor models and may likely be specific to ovarian tumors. Such tumors may also pose a significant challenge in elimination by the T cells; however, NK cells are capable of targeting such tumors and can be exploited to eliminate these tumors in immunotherapeutic strategies.

Efficacy of birinapant in combination with carboplatin in targeting platinum­resistant epithelial ovarian cancers.

Patients diagnosed with epithelial ovarian cancers (EOCs) often suffer from disease relapse associated with the emergence of resistance to standard platinum­based chemotherapy. Treatment of patients with chemo­resistant disease remains a clinical challenge. One mechanism of chemoresistance includes overexpression of pro­survival proteins called inhibitors of apoptosis (IAP) which enable cancer cells to evade apoptosis. Due to their anti­apoptotic activity, association with poor prognosis, and correlation with therapy resistance in multiple malignancies, IAP proteins have become an attractive target for development of anticancer therapeutics. Second mitochondrial activator of caspase (SMAC) mimetics are the most widely used IAP antagonists currently being tested in clinical trials as a monotherapy and in combination with different chemotherapeutic drugs to target different types of cancer. In the present study, the antitumor efficacy of combination therapy with birinapant, a bivalent SMAC mimetic compound, and carboplatin to target platinum­resistant EOC cells was investigated. A 3D organoid bioassay was utilized to test the efficacy of the combination therapy in a panel of 7 EOC cell lines and 10 platinum­resistant primary patient tumor samples. Findings from the in vitro studies demonstrated that the birinapant and carboplatin combination was effective in targeting a subset of ovarian cancer cell lines and platinum­resistant primary patient tumor samples. This combination therapy was also effective in vitro and in vivo in targeting a platinum­resistant patient­derived xenograft (PDX) model established from one of the patient tumors tested. Overall, our study demonstrated that birinapant and carboplatin combination could target a subset of platinum­resistant ovarian cancers and also highlights the potential of the 3D organoid bioassay as a preclinical tool to assess the response to chemotherapy or targeted therapies in ovarian cancer.

A Step in the Right Direction: A Prospective Randomized, Controlled Crossover Trial of Autologous Fat Grafting for Rejuvenation of the Heel.

BACKGROUND: The shock-absorbing soft tissues of the heel are composed of dermis and specialized fat pads. Heel fat pad atrophy is common and can be painful and debilitating. In our previous work, autologous fat grafting was effective for treating pain from forefoot fat pad atrophy. OBJECTIVES: The authors hypothesized that autologous fat grafting to the heel would relieve pain and improve function in patients with heel fat pad atrophy. METHODS: Patients with heel fat pad atrophy and associated pain were recruited and randomized into 2 groups. Group 1 received autologous fat grafting on enrollment and was followed for 2 years. Group 2 received offloading and activity modification for 1 year, then crossed over, underwent autologous fat grafting, and was followed for 1 year afterward. Outcome measures included ultrasound-measured fat pad and dermal thickness; pedobarograph-measured foot pressures and forces; and patient-reported outcomes as measured by the Manchester Foot Pain and Disability Index. RESULTS: Thirteen patients met the inclusion criteria and completed the study. Seven (12 affected feet) were randomized into Group 1; and 6 (9 affected feet) were randomized into Group 2. The average age was 55 years and BMI was 30.5 kg/m2. Demographics did not significantly differ between groups. Heel fat pad thickness increased after autologous fat grafting but returned to baseline at 6 months. However, autologous fat grafting increased dermal thickness significantly and also increased fat pad thickness under a compressive load compared with controls at 6 and 12 months. Foot pain, function, and appearance were also significantly improved compared with controls at 6 and 12 months. CONCLUSIONS: Autologous fat grafting improved patient-reported foot pain, function, and appearance and may rejuvenate local soft tissues in patients with heel fat pad atrophy.

Marginal Mandibular Nerve Mapping Prior to Nonablative Radiofrequency Skin Tightening.

BACKGROUND: Aging skin and increased skin laxity is a prevalent concern of patients. Nonsurgical treatments, such as radiofrequency, are increasing in popularity due to decreased pain, downtime, and scarring. ThermiRF (Thermi, Irving, TX) is a subdermal radiofrequency treatment for tightening skin. When applying radiofrequency treatments to the neck, it is important to avoid ablating the marginal mandibular nerve and causing nerve trauma. OBJECTIVES: The purpose of this study was to locate and record the position of the marginal mandibular nerve in 72 patients undergoing subdermal radiofrequency skin tightening, to determine how often the nerve correlates to its textbook anatomic position. METHODS: Marginal mandibular nerves were located with a nerve stimulator and marked with the subject in both upright and recumbent positions. Photographs were taken and the nerve position in relation to the mandible was recorded. RESULTS: The marginal mandibular nerve was in its correct anatomic position above the mandible in 18% of patients. Nerve position did not shift between the upright and recumbent positions. Only 10% of patients had left-right nerve symmetry. CONCLUSIONS: To avoid nerve injuries, nerve mapping prior to nonablative radiofrequency treatment is recommended. The marginal mandibular nerve is not always in its correct anatomic position or symmetric to the opposing side. Its location cannot be assumed from the textbook anatomic position or from a single-side mapping.

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 µm2) compared to autograft (4.62 ± 3.99 µm2) and PCL/Empty (4.52 ± 5.16 µm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.

Changes in Nerve Conduction Studies After Distal Radius Fracture Fixation Using a Volar Approach and Locked Plate.

Purpose: To determine whether there are changes in nerve conduction studies (NCS) of the median nerve after distal radius fracture (DRF) and to determine how operative fixation through a volar approach with a locking plate contributes to nerve conduction changes. We hypothesized that a considerable percentage of patients would have electrodiagnostic evidence of median neuropathy at the wrist after fracture, but fixation with a volar locked plate would not worsen the electrodiagnostic findings. Methods: This was a prospective cohort study of 14 neurologically asymptomatic patients who underwent surgical treatment of an isolated DRF using a volar plate. All patients underwent surgery within 2 weeks of injury. On the day of surgery and at the 6-week follow-up, patients were clinically examined, Quick-Disabilities of the Arm, Shoulder, and Hand questionnaire was completed, and patients underwent NCS using a handheld device with the unaffected limb, which was used as a comparison. Preoperative and postoperative nerve function were compared with the unaffected limb as a baseline. Results: Patients without symptoms after DRF had a 28% incidence of prolonged latencies compared with reference values for the device used. Distal sensory latencies of the median nerve were 3.64 ± 0.32 ms in the unaffected arm, 3.76 ± 0.70 ms before surgery, and 3.81 ± 0.52 ms after surgery. Distal motor latencies of the median nerve were 3.91 ± 0.59, 3.60 ± 0.68, and 3.88 ± 0.36 ms in respective arms and time points. Quick-Disabilities of the Arm, Shoulder, and Hand scores improved from 77 before surgery to 46 at 6 weeks. Conclusions: Asymptomatic patients may satisfy nerve conduction criteria for median neuropathy at the wrist after DRF; however, open reduction and treatment with a volar locked plate has no significant effect on NCS findings. Type of study/level of evidence: Prognostic II.

The Architecture of Fat Grafting II: Impact of Cannula Diameter.

BACKGROUND: Fat grafting has emerged as the treatment of choice for soft-tissue augmentation and reconstruction. Variability of volume retention remains the greatest challenge for this technique, often requiring multiple operations to achieve the desired volume. Graft that is placed greater than 2 mm from the recipient bed will undergo necrosis. Improved understanding of the architecture of fat within the recipient bed is paramount to improving outcomes. The impact of cannula diameter on graft architecture is unknown. METHODS: Fat was harvested by liposuction and stained with methylene blue. Stained fat was grafted into 4 × 2 × 1-cm sections of excised abdominal tissue with 12-, 14-, 16-, and 19-gauge Coleman cannulas at three different volumes: 0.1, 0.5, and 1.0 cc. Each tissue block was sectioned for stained graft visualization. The diameter of each deposit and percentage with a radius greater than 2 mm were recorded. RESULTS: With an injection volume of 0.1 cc, no fat deposits had a radius greater than 2 mm, regardless of cannula size. A graft volume of 0.5 cc created globules greater than 2 mm with larger cannulas (0 percent with 19-gauge, 2.9 percent with 16-gauge, 6.1 percent with 14-gauge, and 4.3 percent with 12-gauge). Injecting 1.0 cc resulted in a significant increase in the percentage of fat parcels expected to undergo central necrosis (16 percent with 19-gauge, 21 percent with 16-gauge, 26 percent with 14-gauge, and 44 percent with 12-gauge). CONCLUSIONS: Injection cannulas of 14-gauge or larger are more likely to create deposits with dimensions that may be susceptible to central necrosis when injecting 1.0 cc per pass. Smaller cannula sizes or lower volumes per pass should be considered. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Adipose stem cells enhance excisional wound healing in a porcine model.

BACKGROUND: Adipose-derived stem cells (ASCs) are capable of secreting regenerative growth factors and replacing multiple tissue types. Although current literature suggests that ASCs accelerate wound healing and reduce scarring, the dose-response relationship has not been adequately investigated in large animals. We sought to establish a porcine model to optimize dose and delivery. METHODS: Four-centimeter circular, full thickness excisional wounds were created on the backs of Yorkshire pigs. Fluorescently labeled allogeneic porcine ASCs were injected into the superficial wound bed and around the wound perimeter at high (3.0 × 106 cells/cm2; n = 8), medium (1.0 × 106 cells/cm2; n = 8), and low (0.3 × 106 cells/cm2; n = 8) doses. Control wounds received saline injections (n = 8) or no treatment (n = 8). Dressings were changed twice per week, and wound closure was tracked by surface area tracing. Animals were sacrificed at 1 and 2 wk. Wounds were harvested for real-time quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and ASC tracking. RESULTS: Labeled ASCs integrated into treated wounds by 1 wk in a dose-dependent fashion. Epithelial coverage was achieved by 14 d in all wounds. Wounds receiving high-dose ASCs exhibited thicker granulating neodermis at 7 d and greater wound contraction at 14 d. real-time quantitative reverse transcriptase polymerase chain reaction revealed improved collagen 1:collagen 3 (Col1:Col3) ratio in the medium-dose group and enhanced α-smooth muscle actin in the high-dose group at 14 d. Western blot demonstrated increased cluster of differentiation 31 protein at 2 wk in wounds receiving >106 cells/cm2. CONCLUSIONS: Doses up to 3.0 × 106 cells/cm2 were well-tolerated. High-dose ASCs accelerate wound contraction, enhance neovascularization, and may improve scar quality in excisional wounds healing by secondary intention. Doses greater than those previously used may be necessary to achieve desired effects.

Prediction of Treatment Outcomes for Neck Rejuvenation Utilizing a Unique Classification System of Treatment Approach Using a 1440-nm Side-Firing Laser.

BACKGROUND: The appearance of a youthful neck is lost with age causing excessive skin laxity, a loss of subcutaneous fat, prominence of platysmal banding, and jowling. In view of the success obtained with laser treatment for neck rejuvenation, the authors have recently taken an algorithmic approach to developing a 7-category classification system of the aging conditions throughout the anatomic spectrum of three areas: skin, fat, and muscle. This system will correlate with specific treatment options. OBJECTIVE: The objective of the study was to confirm the 7-category classification system and treatment approaches based on clinical outcome data for treatment of the mandibular and submandibular areas, specifically for skin tightening and laser lipolysis after a single 1440-nm laser treatment. METHODS: Patients were treated with a single treatment of PrecisionTX™ 1440-nm wavelength laser on their necks. Baseline and posttreatment photographs were taken and evaluated by 3 blinded reviewers using the Cervicomental Angle Scale (CAS). RESULTS: Subjects were rated grades II-III (2.9 ± 0.8) on average at baseline and grades I-II (1.3 ± 0.5) at follow-up. The average improvement was a mean score of 1.5 ± 0.07. Patients, 23/25 (92%), showed at least a 1 score improvement. CONCLUSIONS: This study confirms a new minimally invasive treatment approach based on a unique classification system with no adverse events reported and high patient satisfaction.

Delivery of adipose-derived stem cells in poloxamer hydrogel improves peripheral nerve regeneration.

INTRODUCTION: Peripheral nerve damage is associated with high long-term morbidity. Because of beneficial secretome, immunomodulatory effects, and ease of clinical translation, transplantation with adipose-derived stem cells (ASC) represents a promising therapeutic modality. METHODS: Effect of ASC delivery in poloxamer hydrogel was assessed in a rat sciatic nerve model of critical-sized (1.5 cm) peripheral nerve injury. Nerve/muscle unit regeneration was assessed via immunostaining explanted nerve, quantitative polymerase chain reaction (qPCR), and histological analysis of reinnervating gastrocnemius muscle. RESULTS: On the basis of viability data, 10% poloxamer hydrogel was selected for in vivo study. Six weeks after transection and repair, the group treated with poloxamer delivered ASCs demonstrated longest axonal regrowth. The qPCR results indicated that the inclusion of ASCs appeared to result in expression of factors that aid in reinnervating muscle tissue. DISCUSSION: Delivery of ASCs in poloxamer addresses multiple facets of the complexity of nerve/muscle unit regeneration, representing a promising avenue for further study. Muscle Nerve 58: 251-260, 2018.

Optimization and Standardization of the Immunodeficient Mouse Model for Assessing Fat Grafting Outcomes.

BACKGROUND: Animal models are often used to assess interventions that might improve fat grafting outcomes; however, there is great variability in the models. The authors sought to determine the predictive value of the immunocompromised mouse model for fat grafting so that experiments could be standardized and optimized. METHODS: Human lipoaspirate injections at different volumes and time points were assessed in a nude mouse model and compared with control injections of nonviable fat. Volume retention and explant histologic score were compared. In a separate study, interanimal reproducibility was determined by implanting a highly consistent hydrogel and measuring variability in volume retention. RESULTS: Injection volume significantly affects adipose resorption kinetics at 6 and 12 weeks. Masson trichrome staining revealed that macrophages were unable to infiltrate large (1 ml) grafts, and oil cysts were not absorbed by 18 weeks, which interfered with interpretation of volume retention data. Nonviable tissue was resorbed when grafts were 0.3 ml, and quantification of graft histologic viability correlated well with graft retention at all study time points. Interanimal variability was measured to be 8.44 percent of the mean retention volume for small graft volumes. CONCLUSIONS: Human fat graft retention in the immunodeficient mouse correlates with graft viability in small, 0.3-ml-volume grafts. However, centralized oil cysts in nonviable 1.0-ml grafts were not resorbed by 18 weeks and thus volume measurements were confounded and not significantly different from viable samples. In addition, tissue injury scores increased in initially healthy fat grafts at 18 weeks, possibly because of a delayed immune reaction.

Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5.

Subsurface Laser and Radiofrequency for Face and Body Rejuvenation.

Minimally invasive devices are a departure from standard laser therapies, because energy is delivered directly below the skin through a 1-mm incision. Lasers can affect such tissues as fat for enhanced disruption, coagulation of small blood vessels, and skin tightening at the right temperatures. Minimally invasive radiofrequency devices can tighten skin but can also improve neck muscle laxity. These devices can achieve results not possible with traditional external devices and, because the skin is not penetrated with energy, a much improved healing profile is seen as well.

Gold Nanoparticle-assisted Selective Photothermolysis of Adipose Tissue (NanoLipo).

BACKGROUND: Conventional suction-assisted lipectomy (SAL) often results in contour irregularity. Selective photothermal heating of adipose tissue by polymer-coated gold nanorods energized by an external near-infrared exposure at 800 nm is introduced in this work to facilitate fat removal. METHODS: The effects of NanoLipo were examined in food-grade porcine abdominal tissue (skin, fat, and fascia) by histology. The efficacy of NanoLipo was compared with that of conventional SAL in vivo in Yucatan mini pigs by quantification of removed subcutaneous tissue and fatty acids and ultrasound measurement of adipose layer thickness. RESULTS: NanoLipo led to the appearance of disruptions in adipose tissue that were not apparent in control groups in ex vivo samples. NanoLipo allowed removal of more subcutaneous tissue (~33% vs ~25% of removed material, P < 0.05) and approximately twice as much free fatty acids (~60% vs ~30% of removed tissue, P < 0.05) in comparison with conventional SAL. Most importantly, NanoLipo led to a greater decrease in adipose layer thickness at 1 month post surgery (P < 0.001). CONCLUSIONS: NanoLipo facilitates removal of a greater quantity of fat and requires less suction time (4 vs 10 minutes) than conventional SAL. As the safety of poly(ethylene-glycol)-coated gold nanorods is well-established, a clinical trial is currently being organized.