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Background Introduction of gut flora into the biliary system is common owing to biliary stenting in patients with obstructing pancreatic head cancer. We hypothesize that alteration of biliary microbiome modifies bile content that modulates pancreatic cancer cell survival. Methods Human bile samples were collected during pancreaticoduodenectomy. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S ribosomal RNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, Panc1) were treated for 24 hours with sterile (nonstented) bile, contaminated (stented) bile, and sterile bile preincubated with 106 colony forming unit of live bacteria isolated from contaminated bile or a panel of bile acids for 24 hours at 37°C, and evaluated using CellTiter-Blue Cell Viability Assay (Promega Corp. Madison, WI). Human bile (30–50 μl/mouse) was coinjected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice to evaluate the impact of bile on peritoneal metastasis 3 to 4 weeks after tumor challenge. Results While all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had diminished antitumor effect. All sterile bile (n = 4) reduced pancreatic cancer cell survival in vitro. Only 40% (2/5) of contaminated bile samples had significant effect. Preincubation of sterile bile with live Enterococcus faecalis or Streptococcus oralis modified the antitumor effect of sterile bile. These changes were not observed with culture media preincubated with live bacteria, suggesting live gut bacteria can modify the antitumor components present in bile. Conjugated bile acids were more potent than unconjugated cholic acid in reducing pancreatic cancer cell survival. Conclusion Alteration of bile microbiome from biliary stenting has a direct impact on pancreatic cancer cell survival. Further study is warranted to determine if this microbiome shift alters tumor microenvironment.

BACKGROUND: Disease burden in patients with peritoneal carcinomatosis (PC) is difficult to estimate. We evaluated whether peritoneal cell-free tumor DNA can be used as a measure of disease burden. METHODS: Malignant ascites or peritoneal lavage fluids were collected from patients with PC under approved IRB protocol. Cell-free DNA was extracted from peritoneal fluid. Droplet digital PCR (ddPCR) was performed using a commercially available KRAS G12/G13 screening kit. Mutant allele frequency (MAF) was calculated based on the numbers of KRAS wild-type and mutant droplets. Clinicopathological, treatment and outcome data were abstracted and correlated with MAF of cell-free KRAS mutant DNA. RESULTS: Cell-free KRAS mutant DNA was detected in 15/37 (40%) malignant peritoneal fluids with a MAF of 0.1% to 26.2%. While peritoneal cell-free KRAS mutant DNA was detected in all the patients with KRAS mutant tumors (N=10), 3/16 (19%) patients with KRAS wild-type tumors also had peritoneal cell-free KRAS mutant DNA. We also found that 71% (5/7) of patients with disease amenable to cytoreductive surgery (CRS) had a MAF of < 1% (median: 0.5%, range: 0.1-4.7%), while 75% (6/8) of patients with unresectable disease had a MAF of > 1% (median: 4.4%, range: 0.1-26.2%). CONCLUSIONS: This pilot proof-of-principle study suggests that peritoneal cell-free tumor DNA detected by ddPCR may enable prediction of disease burden and a measure of disease amenable to CRS in patients with PC.

BACKGROUND. The treatment options for patients with peritoneal carcinomatosis (PC) of gastrointestinal and pancreaticobiliary origins are limited. The virus-like particle, CMP-001, composed of the Qβ bacteriophage capsid protein encapsulating a CpG-A oligodeoxynucleotide, activates plasmacytoid dendritic cells (pDCs) and triggers interferon alpha (IFNα) release, leading to a cascade of anti-tumor immune effects. METHODS. To evaluate the ability of CMP-001 to trigger an immune response in patients with PC, peritoneal cells were isolated and stimulated ex vivo with CMP-001. Both IFNα release and percentage of pDC were quantified using enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. To evaluate the anti-tumor response in vivo, murine PC models were generated using mouse cancer cell lines (Panc02 and MC38) in immunocompetent mice treated with intraperitoneal CMP-001 or saline control. Survival was followed, and the immunophenotype of cells in the peritoneal tumor microenvironment was evaluated. RESULTS. The pDCs accounted for 1% (range 0.1–3.9%; n = 17) of the isolated peritoneal cells. Ex vivo CMP-001 stimulation of the peritoneal cells released an average of 0.77 ng/ml of IFNα (range, 0–4700 pg/ml; n = 14). The IFNα concentration was proportional to the percentage of pDCs present in the peritoneal cell mixture (r = 0.6; p = 0.037). In murine PC models, intraperitoneal CMP-001 treatment elicited an anti-tumor immune response including an increase in chemokines (RANTES and MIP-1β), pro-inflammatory cytokines (IFNγ, interleukin 6 [IL-6], and IL-12), and peritoneal/tumor immune infiltration (CD4(+)/CD8(+) T and natural killer [NK] cells). The CMP-001 treatment improved survival in both the Panc02 (median, 35 vs 28 days) and the MC38 (median: 57 vs 35 days) PC models (p < 0.05). CONCLUSIONS. As a novel immunotherapeutic agent, CMP-001 may be effective for treating patients with PC.

Therapy for lung cancer patients includes surgery, radiation, and chemotherapy. While treatments initially illicit desirable responses, the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer versus normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) is selectively toxic to lung cancer cells versus normal bronchial epithelial cells. Furthermore, cancer cell toxicity is exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu can be attributed to its differential Cu ionophore capabilities. DSF/Cu treatment significantly increased total cellular Cu levels, with higher Cu present in tumor versus normal cells and in cancer cells at 1% O2 versus 21% O2. DSF toxicity was shown to be dependent on Cu by inhibiting toxicity with a Cu chelator, bathocuproinedisulfonic acid (BCS), which inhibits redox cycling. Toxicity was also shown to be dependent on oxidative stress mechanisms that are initiated by Cu, including the production of superoxide and peroxide, which were inhibited by a superoxide dismutase (SOD) mimetic, GC4419, and overexpression of catalase. DSF/Cu treatment also induced lipid oxidation, increased mitochondrial superoxide production, and decreased mitochondrial membrane potential, which were detected by C11-BODIPY, MitoSox, and JC-1. In addition to its selective toxicity to hypoxic cancer cells, DSF/Cu also enhanced radio-chemotherapy-induced cancer cell killing and reduced therapy resistance at hypoxia. DSF also decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF is a promising adjuvant for cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

Objectives Renal cell carcinoma (RCC) is an immunogenic tumor, and multiple immunostimulatory therapies are in use or under development for patients with inoperable tumors. However, a major drawback to the use of immunotherapy for RCC is that renal tumors are also immunosuppressive. As a result, current immunotherapies are curative in Methods and materials Peripheral blood samples were taken from 53 consented subjects with renal masses before cytoreductive nephrectomy and again at clinic visits approximately 30 days after nephrectomy. Samples were also obtained from 10 healthy age- and gender-matched controls. Blood samples from clear cell RCC subjects were analyzed by multi-parameter flow cytometry to determine leukocyte subset composition and multiplex array to evaluate plasma proteins. Results Pre-nephrectomy, clear cell tumors were associated with systemic accumulations of both “exhausted” CD8+ T cells, as indicated by surface BTLA expression, and monocytic CD14 + HLA-DR neg CD33 + myeloid-derived suppressor cells (MDSC). Subjects with T3 clear cell RCC also had a unique pro-tumorigenic and inflammatory cytokine/chemokine profile characterized by high serum concentrations of IL-1β, IL-2, IL-5, IL-7, IL-8, IL-17, TNF-α, MCP-1 and MIP-1β. At an early post-nephrectomy time point (~30 d), we found the systemic immune response to be largely unaltered. The only significant change was a decrease in the mean percentage of circulating BTLA + CD8 + T cells. All other cellular and soluble immune parameters we examined were unaltered by the removal of the primary tumor. Conclusions In the first month following surgery, nephrectomy may relieve systemic CD8 T cell exhaustion marked by BTLA expression, but continuing inflammation and MDSC presence likely counteract this positive effect. Future determination of how this systemic immune signature becomes altered during metastatic progression could provide novel targets for neoadjuvant immunotherapy in RCC.

Peritoneal carcinomatosis is a common form of metastasis occurring in approximately 10% of gastrointestinal and pancreatic cancers with life expectancy often less than 6-12 months. Due to the limited options for treatment of peritoneal carcinomatosis, there is a significant need for novel therapeutic strategies. We explored the therapeutic potential of CMP-001, a novel virus-like particle composed of the Qβ bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide (CpG-A ODN) in this cancer. CpG-A ODN is a known activator of toll-like receptor 9 (TLR9), which is expressed by human and murine plasmacytoid dendritic cells (pDCs). Initial studies evaluated the effect of CMP-001 in vitro on cells obtained from the ascitic fluid of patients with peritoneal carcinomatosis. These studies demonstrated that pDCs are present in the ascitic fluid and produce IFN-α in response to CMP-001 stimulation. A mouse model of peritoneal carcinomatosis was used to further explore the therapeutic potential of CMP-001. C57BL/6 mice were challenged intraperitoneally (i.p.) with Panc02 mouse pancreatic cancer cells. On days 5, 9, and 13 post-tumor challenge mice were treated i.p. with either saline or 100 μg CMP-001. Mice treated with CMP-001 had a median survival of 35 days compared to mice treated with saline alone with a median survival of 28 days (n = 10 per group, p = 0.028). Examination of the immune response demonstrated CMP-001 induced an influx of dendritic cells (CMP- 001 8.99% ± 0.95 vs saline 4.57% ± 0.7, p = 0.0015), NK cells (CMP-001 0.26% ± 0.052 vs saline 0.087% ± 0.0085, p = 0.006), and CD4+ T cells (CMP-001 3.29% ± 0.85 vs saline 0.95% ± 0.097, p = 0.0168) into the ascitic fluid. In addition, CMP-001 induced a significant increase in antigen-experienced, effector and memory, CD11ahiCD44hi CD4+ T cells (CMP-001 54.55% ± 6.52 vs saline 28.12% ± 3.68, p = 0.0028) and CD11ahiCD8αlo CD8+ T cells (CMP-001 58.49% ± 4.47 vs saline 23.04% ± 2.86, p < 0.0001) in the ascites. These data demonstrate that CMP-001 treatment activates pDCs in the peritoneal fluid of patients with carcinomatosis. In addition, CMP-001 stimulates a robust immune response in the peritoneal cavity of mice with peritoneal carcinomatosis. This robust immune response likely contributes to the enhanced survival and decreased disease progression in these mice. Collectively, these promising preclinical results suggest that CMP-001 may have potential as an immunotherapy for the treatment of patients with peritoneal carcinomatosis and is worthy of further evaluation. Citation Format: Ann M. Miller, Caitlin Lemke-Miltner, Sue Blackwell, Ann Tomanek-Chalkley, Kristen Coleman, George Weiner, Carlos Chan. CMP-001, a virus-like particle containing immunostimulatory CpG-A, for treatment of peritoneal carcinomatosis of gastrointestinal and pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3273.

Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM) but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266), using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG) induced ER stress as detected by qPCR (BiP, ATF4) and immunoblotting (BiP, CHOP) and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM cells via 2-DG and 10-TPP combination therapy.

Obesity is a mounting health concern in the United States and is associated with an increased risk for developing several cancers, including renal cell carcinoma (RCC). Despite this, little is known regarding the impact of obesity on antitumor immunity. Because dendritic cells (DC) are critical regulators of antitumor immunity, we examined the combined effects of obesity and tumor outgrowth on DC function. Using a diet-induced obesity (DIO) model, DC function was evaluated in mice bearing orthotopic RCC and in tumor-free controls. Tumor-free DIO mice had profoundly altered serum cytokine and chemokine profiles, with upregulation of 15 proteins, including IL-1α, IL-17, and LIF. Tumor-free DIO mice had elevated percentages of conventional splenic DC that were impaired in their ability to stimulate naive T cell expansion, although they were phenotypically similar to normal weight (NW) controls. In DIO mice, intrarenal RCC tumor challenge in the absence of therapy led to increased local infiltration by T cell-suppressive DC and accelerated early tumor outgrowth. Following administration of a DC-dependent immunotherapy, established RCC tumors regressed in normal weight mice. The same immunotherapy was ineffective in DIO mice and was characterized by an accumulation of regulatory DC in tumor-bearing kidneys, decreased local infiltration by IFN-γ–producing CD8 T cells, and progressive tumor outgrowth. Our results suggest that the presence of obesity as a comorbidity can impair the efficacy of DC-dependent antitumor immunotherapies.

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 h after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir-145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir-145 expression. Decitabine-treated keratinocytes also show increased mir-302c and proliferation similar to other Oct4(+) cells. Treatment with doxorubicin, another cancer drug, induces expression of mir-145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co-treatment with decitabine and doxorubicin results first in increased OCT4 and mir-145, then a decrease in both, suggesting that OCT4 and mir-145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4(+) cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

Cancer cells have increased steady state levels of reactive oxygen species (ROS; O2•- and H2O2) compared to normal cells. It has been proposed that using redox active agents that further increase ROS levels will result is selective cancer cell death. This metabolic frailty can be targeted using drugs deemed safe for human use, ascorbate (ASC) and disulfiram (DSF), via a mechanism of H2O2 production. CuATSM is a drug being used in clinical trials to treat ALS disease. Imaging studies have shown that CuATSM preferentially concentrates in hypoxic tissues, releasing its Cu after entering cells. Copper (Cu) can participate in oxidation reactions that result in highly toxic hydroxyl radicals. Tumors often have areas of hypoxic tissue that exhibit resistance to ionizing radiation (IR). Our hypothesis is that CuATSM can be used to sensitize hypoxic regions of tumors to IR, ASC and/or DSF. Method: H2O2 flux after addition of ASC or DSF was measured in small cell lung cancer (SCLC) cells DMS53 using the 3-aminotriazole method. The Cu uptake of these cells was measured in normoxia and hypoxia after treatment with CuATSM. DMS53 and DMS273 (SCLC lines) were treated with ASC, CuATSM, and/or DSF+CuSO4 at varying oxygen tensions with and without IR and clonogenic assays were performed. Results: Pharmacologically relevant dosing of DSF and ASC resulted in significantly higher fluxes of H2O2 compared to control. Both DSF and ASC enhanced IR clonogenic cell death in SCLC lines. Treatment with CuATSM resulted in increased intracellular Cu and enhanced cell death from ASC and IR in hypoxic conditions. Conclusion: These observations support the hypothesis that the differences in steady-state level of ROS in small cell lung cancer cells can be exploited to develop effective therapies using ASC and DSF. Furthermore CuATSM can enhance responses in hypoxic tumor tissues to both IR and ASC. (Supported by P50 CA174521, T32 CA078586, P30 CA086862 and Carver Research Program of Excellence in Redox Biology and Medicine.) Citation Format: Sebastian Sciegienka, Samuel Rodman, Ann Tomanek-Chalkley, Kelly C. Falls, Douglas R. Spitz, Melissa A. Fath. Sensitizing hypoxic small cell lung cancer cells to radiation and hydrogen peroxide-producing agents using CuATSM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3219.

To test the influence of fibroblasts on epithelial morphology and expression of keratinocyte proteins and barrier lipids, we bioengineered homotypic and heterotypic oral mucosae and skin using cultured adult human cells. Fibroblasts were allowed to modify collagen type I gels for 2 weeks before keratinocytes were added. The organotypic cultures were then grown at the air-liquid interface for 4 weeks. In homotypic combinations, epithelial morphology and protein expression closely mimicked those in vivo. In heterotypic combinations, the morphology resembled that in vivo and keratinocytes expressed their typical markers, except when skin keratinocytes were recombined with alveolar fibroblasts; they expressed K19, K4, and K13, which is similar to oral mucosal epithelia rather than to the epidermis. Morphologically, the stratum corneum layers were typical for the epithelial tissues. Grafting the bioengineered cultures to the backs of Nude mice did not change the results, suggesting that our findings are not merely a culture phenomenon. Lipid profiles of the homotypic combinations mimicked the profiles found in the normal epithelial tissues, except that the engineered alveolar epithelium expressed more ceramide 2 than that in vivo. In the heterotypic combinations, keratinocytes appeared to control the lipid profile, except in the combination of skin keratinocytes with alveolar fibroblasts, wherein the ceramide profile appeared to be partly that of alveolar epithelium and partly that of epidermis. These results suggest that cultured adult fibroblasts and keratinocytes are sufficient to recapitulate graftable oral tissues, and, except for alveolar fibroblasts, the type of fibroblast had little influence on keratinocyte differentiation.

Renal cell carcinoma (RCC) is an immunogenic tumor, and multiple immunostimulatory therapies are in use or under development for patients with inoperable tumors. However, a major drawback to the use of immunotherapy for RCC is that renal tumors are also immunosuppressive. As a result, current immunotherapies are curative in10% of patients with RCC. To better understand the systemic immune response to RCC, we performed a comprehensive examination of the leukocyte and cytokine/chemokine composition in the peripheral blood of patients with localized clear cell renal tumors pre- and post-nephrectomy.Peripheral blood samples were taken from 53 consented subjects with renal masses before cytoreductive nephrectomy and again at clinic visits approximately 30 days after nephrectomy. Samples were also obtained from 10 healthy age- and gender-matched controls. Blood samples from clear cell RCC subjects were analyzed by multi-parameter flow cytometry to determine leukocyte subset composition and multiplex array to evaluate plasma proteins.Pre-nephrectomy, clear cell tumors were associated with systemic accumulations of both "exhausted" CD8+ T cells, as indicated by surface BTLA expression, and monocytic CD14(+)HLA-DR(neg)CD33(+) myeloid-derived suppressor cells (MDSC). Subjects with T3 clear cell RCC also had a unique pro-tumorigenic and inflammatory cytokine/chemokine profile characterized by high serum concentrations of IL-1β, IL-2, IL-5, IL-7, IL-8, IL-17, TNF-α, MCP-1 and MIP-1β. At an early post-nephrectomy time point (~30 d), we found the systemic immune response to be largely unaltered. The only significant change was a decrease in the mean percentage of circulating BTLA(+)CD8(+) T cells. All other cellular and soluble immune parameters we examined were unaltered by the removal of the primary tumor.In the first month following surgery, nephrectomy may relieve systemic CD8 T cell exhaustion marked by BTLA expression, but continuing inflammation and MDSC presence likely counteract this positive effect. Future determination of how this systemic immune signature becomes altered during metastatic progression could provide novel targets for neoadjuvant immunotherapy in RCC.

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 hours after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir-145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir-145 expression. Decitabine-treated keratinocytes also show increased mir-302c and proliferation similar to other Oct4+ cells. Treatment with doxorubicin, another cancer drug, induces expression of mir-145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co-treatment with decitabine and doxorubicin results first in increased OCT4 and mir-145, then a decrease in both, suggesting that OCT4 and mir-145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4+ cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

In this study, we describe a simple system in which human keratinocytes can be redirected to an alternative differentiation pathway. We transiently transfected freshly isolated human skin keratinocytes with the single transcription factor OCT4. Within 2 days these cells displayed expression of endogenous embryonic genes and showed reduced genomic methylation. More importantly, these cells could be specifically converted into neuronal and contractile mesenchymal cell types. Redirected differentiation was confirmed by expression of neuronal and mesenchymal cell mRNA and protein, and through a functional assay in which the newly differentiated mesenchymal cells contracted collagen gels as efficiently as authentic myofibroblasts. Thus, to generate patient-specific cells for therapeutic purposes, it may not be necessary to completely reprogram somatic cells into induced pluripotent stem cells before altering their differentiation and grafting them into new tissues.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.