Michael P. Kim

Chemoresistant Colorectal Cancer Cells, the Cancer Stem Cell Phenotype, and Increased Sensitivity to Insulin-like Growth Factor-I Receptor Inhibition

5-Fluorouracil (5FU) and oxaliplatin are standard therapy for metastatic colorectal cancer (CRC), but the development of chemoresistance is inevitable. Because cancer stem cells (CSC) are hypothesized to be chemoresistant, we investigated CSC properties in newly developed chemoresistant CRC cell lines and sought to identify targets for therapy. The human CRC cell line HT29 was exposed to increasing doses of 5FU (HT29/5FU-R) or oxaliplatin (HT29/OxR) to achieve resistance at clinically relevant doses. Western blotting and flow cytometry were done to determine molecular alterations. The insulin-like growth factor-I receptor (IGF-IR) monoclonal antibody (mAb) AVE-1642 was used to inhibit signaling in vitro and in vivo using murine xenograft models. HT29/5FU-R and HT29/OxR showed 16- to 30-fold enrichment of CD133(+) cells and 2-fold enrichment of CD44(+) cells (putative CRC CSC markers). Resistant cells were enriched 5- to 22-fold for double-positive (CD133(+)/CD44(+)) cells. Consistent with the CSC phenotype, resistant cells exhibited a decrease in cellular proliferation in vitro (47-59%; P < 0.05). Phosphorylated and total IGF-IR levels were increased in resistant cell lines. HT29/5FU-R and HT29/OxR cells were approximately 5-fold more responsive to IGF-IR inhibition relative to parental cells (P < 0.01) in vitro. Tumors derived from HT29/OxR cells showed significantly greater growth inhibition in response to an IGF-IR mAb than did parental cells (P < 0.05). Chemoresistant CRC cells are enriched for CSC markers and the CSC phenotype. Chemotherapy-induced IGF-IR activation provided for enhanced sensitivity to IGF-IR-targeted therapy. Identification of CSC targets presents a novel therapeutic approach in this disease.

ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma

Background Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends. Methodology/Principal Findings We have isolated populations of cells with high ALDH activity (ALDHhigh) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDHhigh and ALDHlow cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice. Conclusions/Significance We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133+ and ALDHlow cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDHhigh cell populations. ALDHhigh/CD44+/CD24+ or ALDHlow/CD44+/CD24+ phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.

Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice

For decades, xenografts using well-established human tumor cell lines have been the most commonly used models to study human cancers in mice. More recently, human tumors implanted directly into immunodeficient mice have become increasingly popular as evidence accrues that they more accurately recapitulate features of patient tumors. Here we describe our protocols for the orthotopic and heterotopic implantation of pancreatic cancer cell lines and freshly isolated patient tumors into immunodeficient mice. We also describe procedures for the digestion of tumors into single-cell suspensions for the isolation of subpopulations of tumor cells. Orthotopic or heterotopic implantation of established cell lines requires 1–2 h, with 1-cm tumors arising after 2–5 weeks. Engraftment of patient tumor samples takes ∼2 h and growth of palpable tumor requires ∼14 weeks. Once established, direct xenograft tumors require 2 and 5 h for heterotopic and orthotopic implantation, respectively, and 5–6 weeks for palpable tumor growth.

Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells Is Mediated by Oxidative Stress

Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the nonreceptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We show that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin-resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single-agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations.

Gemcitabine Resistance in Pancreatic Cancer: Picking the Key Players

The diagnosis of pancreatic cancer confers a grim prognosis that has changed little over the last 30 years. Due to the lack of any efficacious screening or biomarkers for pancreatic cancer, presentation with advanced local disease is typical (classically, painless jaundice) and morbidity aside, 4%

Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis.

Src family kinases (SFKs) are signaling enzymes that have long been recognized to regulate critical cellular processes such as proliferation, survival, migration, and metastasis. Recently, considerable work has elucidated mechanisms by which SFKs regulate normal and pathologic processes in vascular biology, including endothelial cell proliferation and permeability. Further, when inappropriately activated, SFKs promote pathologic inflammatory processes and tumor metastasis, in part through their effects on the regulation of endothelial monolayer permeability. In this review, we discuss the roles of aberrantly activated SFKs in mediating endothelial permeability in the context of inflammatory states and tumor cell metastasis. We further summarize recent efforts to translate Src-specific inhibitors into therapy for systemic inflammatory conditions and numerous solid organ cancers.

Preoperative Therapy and Pancreatoduodenectomy for Pancreatic Ductal Adenocarcinoma: a 25-Year Single-Institution Experience

BackgroundThe purpose of this study was to evaluate a single-institution experience with delivery of preoperative therapy to patients with pancreatic ductal adenocarcinoma (PDAC) prior to pancreatoduodenectomy (PD).MethodsConsecutive patients (622) with PDAC who underwent PD following chemotherapy and/or chemoradiation between 1990 and 2014 were retrospectively reviewed. Preoperative treatment regimens, clinicopathologic characteristics, operative details, and long-term outcomes in four successive time periods (1990–1999, 2000–2004, 2005–2009, 2010–2014) were evaluated and compared. ResultsThe average number of patients per year who underwent PD following preoperative therapy as well as the proportion of operations performed for borderline resectable and locally advanced (BR/LA) tumors increased over time. The use of induction systemic chemotherapy, as well as postoperative adjuvant chemotherapy, also increased over time. Throughout the study period, the mean EBL decreased while R0 margin rates and vascular resection rates increased overall. Despite the increase in BR/LA resections, locoregional recurrence (LR) rates remained similar over time, and overall survival (OS) improved significantly (median 24.1, 28.1, 37.3, 43.4 months, respectively, p < 0.0001).ConclusionsDespite increases in case complexity, relatively low rates of LR have been maintained while significant improvements in OS have been observed. Further improvements in patient outcomes will likely require disruptive advances in systemic therapy.

Association of Clinical Factors With a Major Pathologic Response Following Preoperative Therapy for Pancreatic Ductal Adenocarcinoma

Importance We previously demonstrated that a major pathologic response to preoperative therapy, defined histopathologically by the presence of less than 5% viable cancer cells in the surgical specimen, is an important prognostic factor for patients with pancreatic ductal adenocarcinoma. However, to our knowledge, the patients most likely to experience a significant response to therapy are undefined. Objective To identify clinical factors associated with major pathologic response in a large cohort of patients who underwent preoperative therapy and pancreatectomy for pancreatic ductal adenocarcinoma. Design, Setting, and Participants Retrospective review of a prospectively maintained database at University of Texas MD Anderson Cancer Center. The study included 583 patients with histopathologically confirmed pancreatic ductal adenocarcinoma who received preoperative therapy prior to pancreatectomy between 1990 and 2015. Exposures Preoperative therapy consisted of systemic chemotherapy alone (n = 38; 6.5%), chemoradiation alone (n = 261; 44.8%), or both (n = 284; 48.7%) prior to pancreatoduodenectomy (n = 514; 88.2%), distal pancreatectomy (n = 62; 10.6%), or total pancreatectomy (n = 7; 1.2%). Main Outcomes and Measures Clinical variables associated with a major pathologic response (pathologic complete response or <5% residual cancer cells) were evaluated using logistic regression. Results Among all patients, the mean (SD) age was 63.7 (9.2) years, and 53.0% were men. A major pathologic response was seen in 77 patients (13.2%) including 23 (3.9%) who had a complete pathologic response. The median overall survival duration was significantly longer for patients who had a major response than for those who did not (73.4 months vs 32.2 months, P < .001). On multivariate logistic regression, only age younger than 50 years, baseline serum cancer antigen 19-9 level less than 200 U/mL, and gemcitabine as a radiosensitizer were associated with a major response. The number of these positive factors was associated with the likelihood of a major response in a stepwise fashion (0, 7.5%; 1, 12.7%; 2, 16.9%; 3, 35.7%; P = .009). Conclusions and Relevance Although a major pathologic response occurs infrequently following preoperative therapy for pancreatic ductal adenocarcinoma, it is associated with a significantly improved prognosis. Of the patient- and treatment-related factors we analyzed, only young age, low baseline cancer antigen 19-9, and gemcitabine as a radiosensitizer were associated with a major pathologic response. Given its association with long-term survival, better predictors of response and more effective preoperative regimens should be aggressively sought.

Impact of pancreatectomy on long‐term patient‐reported symptoms and quality of life in recurrence‐free survivors of pancreatic and periampullary neoplasms

Long term patient‐reported symptoms and quality of life (QOL) are important outcome metrics following cancer operations, but have been poorly described in patients who have previously undergone pancreatectomy.

Selective Perioperative Administration of Pasireotide is More Cost-Effective Than Routine Administration for Pancreatic Fistula Prophylaxis

BackgroundIn a randomized trial, pasireotide significantly decreased the incidence and severity of postoperative pancreatic fistula (POPF). Subsequent analyses concluded that its routine use is cost-effective. We hypothesized that selective administration of the drug to patients at high risk for POPF would be more cost-effective.Study DesignConsecutive patients who did not receive pasireotide and underwent pancreatoduodenectomy (PD) or distal pancreatectomy (DP) between July 2011 and January 2014 were distributed into groups based on their risk of POPF using a multivariate recursive partitioning regression tree analysis (RPA) of preoperative clinical factors. The costs of treating hypothetical patients in each risk group were then computed based upon actual institutional hospital costs and previously published relative risk values associated with pasireotide.ResultsAmong 315 patients who underwent pancreatectomy, grade B/C POPF occurred in 64 (20%). RPA allocated patients who underwent PD into four groups with a risk for grade B/C POPF of 0, 10, 29, or 60% (P < 0.001) on the basis of diagnosis, pancreatic duct diameter, and body mass index. Patients who underwent DP were allocated to three groups with a grade B/C POPF risk of 14, 26, or 44% (P = 0.05) on the basis of pancreatic duct diameter alone. Although the routine administration of pasireotide to all 315 patients would have theoretically saved